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Live performances
Madonna has performed "Like a Prayer" on six of her concert tours: Blond Ambition (1990), Re-Invention (2004), Sticky & Sweet (2008―2009), MDNA (2012), Madame X (2019―2020), and Celebration (2023―2024). On the first one, a dance version was performed. The stage was set up to resemble a candlelit cathedral, while Madonna wore black vestments. Carlton Wilborn, one of the dancers, played a priest she interacted with. Reviewing the Houston concert, the staff of the Orlando Sentinel highlighted the number's "joyous gospel and heavy gothic organ scorings". Performances of the song can be found in Blond Ambition Japan Tour 90, taped in Yokohama, Blond Ambition World Tour Live, taped in Nice, and in the documentary Madonna: Truth or Dare (1991).

While promoting her ninth studio album American Life in 2003, Madonna sang "Like a Prayer" at an HMV store in Oxford, and on the MTV special On Stage & On the Record. She then did an acoustic rendition on the Re-Invention World Tour. Named the concert's "best blend of music and visuals" by The Washington Times' Christian Toto, Madonna sang as the backdrops showed a video of black churchgoers and Hebrew letters. A performance of "Like a Prayer" from this tour can be found on the I'm Going to Tell You a Secret live album and documentary (2006). On July 2, 2005, Madonna and a gospel choir sang "Like a Prayer" at the Live 8 London concert. During the performance, she was joined by Birhan Woldu, a woman who appeared as a malnourished child in the documentary on African famine that aired during the 1985 Live Aid concert.

A "thumping" house remix was sung on the Sticky & Sweet Tour. The number had dancers dressed as ninjas, the singer in a silver bustier and a blonde wig, and spiritual imagery and quotes from the Bible on the backdrops. From The Province, Stuart Derdeyn named "Like a Prayer" the concert's "musical highlight in terms of creating that 'stadium' thing. People were up off their feet, clapping along and pretty well losing it". The song's performance was included on the Sticky & Sweet Tour live album release (2010), recorded during the four concerts in Buenos Aires, Argentina.

Madonna sang a stripped down "Like a Prayer" during Hope for Haiti Now, a charity telethon in benefit of the 2010 Haiti earthquake. She sang backed by a choir, with the only non-vocal instrumentation provided by a guitarist and a keyboard player. MTV's Maura Johnston praised the choir for being a "perfect counterpoint to Madonna's vocals". On February 5, 2012, "Like a Prayer" was the closing song of the Super Bowl XLVI halftime show. Madonna sang on top of a raised platform, and was joined by Cee Lo Green and a choir in robes. Reviewing the set, Ken Tucker from Entertainment Weekly felt both singers "soared" during the number. On the MDNA Tour, it was reworked as a "gospel-anthem". Joined by a 36-piece choir, the number had Madonna "on the floor of the front stage, writhing in orgasmic ways", as noted by The Globe and Mail's Brad Wheeler. The performances of the song at the November 19–20 shows in Miami were recorded and released in the singer's fourth live album, MDNA World Tour (2013).

Despite not being part of the official set list, Madonna sang "Like a Prayer" on some dates of 2015―2016's Rebel Heart Tour, including Inglewood, Birmingham, London, and Stockholm. On the latter city, she sang it following a teary speech addressing the Paris attacks. On November 9, 2015, following one of the Paris concerts, the singer gave a surprise impromptu mini-concert at the Place de la République, and again sang "Like a Prayer" as tribute to the victims of the attacks. The song's performance at the March 19–20, 2016 shows in Sydney's Allphones Arena was recorded and released on Madonna's fifth live album, Rebel Heart Tour (2017). On November 7, 2016, Madonna performed the song as part of an impromptu concert at Washington Square Park in support of Hillary Clinton's presidential campaign.



Madonna did a "somber" rendition of "Like a Prayer" at the 2018 Met Gala. Donning a "monastic" cloak and surrounded by dressed as monks, she began the performance by walking down the stairs to the sound of church bells. On January 1st, 2019, she made a surprise appearance at the Stonewall Inn, and sang "Like a Prayer" and Elvis Presley's "Can't Help Falling in Love" (1961), with her son David Banda on guitar. Four months later, Madonna sang a hip-hop-influenced "Like a Prayer" on the finals of the Eurovision Song Contest 2019. Widely criticized for seemingly being a political statement on the Israeli–Palestinian conflict, the number had two dancers brandishing the Palestinian and Israeli flags on their backs and embracing each other. Madonna was further criticized for singing out of tune.

The song's performance on the Madame X Tour had a choir standing on an X-shaped staircase, and the singer wearing a black hooded robe encrusted with crucifixes; the music video was projected on the background as she sang. From Rolling Stone, Rob Sheffield described the number as "sacred yet also sleazy — the ultimate Madonna combination". A performance of "Like a Prayer" was included on both the film that chronicled the tour and its accompanying live album. On October 9, 2021, following the release of the Madame X concert film, Madonna and Jon Batiste led an impromptu sing-along of "Like a Prayer" down Harlem's 126th Street. They were surrounded by a "quartet of backup singers, and a little bit of light security", while the singer, who donned a black cocktail dress with a "dramatic leg slit", lace gloves, and long blonde wig, allowed attendees to "buzz around her like bees celebrating their queen", as noted by W magazine's Kyle Munzenrieder.

A "pumping [and] hypnotic" remix of "Like a Prayer" that included elements of Sam Smith and Kim Petras' "Unholy" (2022), was sung on the Celebration Tour. The number had shirtless dancers doing crucifix poses on top of a rotating carousel adorned with crosses, and a Prince look-alike playing a "beatific" guitar solo. On his review of the concert, Jed Gottlieb from the Boston Herald said the performance featured "one of the most arresting pieces of choreography in any pop show". In 2022, Madonna revealed that "Like a Prayer" is her favorite song to perform live.

Cover versions and usage
One of the first covers of the song was an acoustic rendition done by English folk singer-songwriter John Wesley Harding, for his 1989 extended play God Made Me Do It: The Christmas EP. The 1999 and 2000 compilation albums Virgin Voices: A Tribute To Madonna, Vol. 1 and Vol. 2, included a cover of the track by American singer Loleatta Holloway and German electro-industrial band Bigod 20, respectively. In 2002, French–Dutch group Mad'House did an Eurodance version of "Like a Prayer" that was included on their album Absolutely Mad. It acheived commercial success, reaching the chart's top-five in countries such as Austria, Belgium, Denmark, France, and Germany, and peaking at number two on Billboard European Hot 100 Singles chart.

In 2007, a cover by American singer Lavender Diamond was included on the tribute compilation Through the Wilderness. Three years later, on March 2010, American gothic metal band We Are the Fallen sang "Like a Prayer" at a concert in London. Nick Duerden from Spin complimented the rendition for being "so successfully pulverized that one wondered whether it wasn't written specifically to become the world's greatest heavy rock anthem". The following month, "Like a Prayer" was included on "The Power of Madonna", the fifteenth episode of American television series Glee. The number was named the episode's best by The Guardian's Anna Pickard, who named it the most "Glee-encapsulating performance in ages". The rendition was then included on the extended play Glee: The Music, The Power of Madonna. In the United States, the cover peaked at number 27 on the Billboard Hot 100, and at number ten on the Hot Digital Songs, with sales of 87,000. Overseas, Glee's cover reached number 28 in Australia, number two in Ireland, and number 16 in the United Kingdom.

On June, British DJ Meck and American musician Dino released "Feels Like a Prayer", which was described as a "tweaking" of the original. This release reached number the sixth spot of the Hot Dance Club Songs chart. "Feels Like a Prayer" also reached the top five in the Flanders region of Belgium and the Netherlands, as well as the top twenty in Belgium's Wallon region. In 2013, Canadian singer Nelly Furtado covered "Like a Prayer" on her Spirit Indestructible Tour. Four years later, on June 2017, Leonard released a piano ballad version of the track, with vocals from American singer Dana Williams. That same month, Brazilian singer Luiza Possi covered "Like a Prayer" during the show Domingão do Faustão, in a similar performance to that of the Blond Ambition tour. On July, Possi again sang the song at a concert in tribute to Michael Jackson.

British singer Sophie Ellis-Bextor covered "Like a Prayer" on June 2020 during one of her Kitchen Disco shows —a series of at-home mini-concerts broadcast live through her Instagram account during the COVID-19 pandemic. One year later, on her TV special Miley Cyrus Presents Stand by You, American singer Miley Cyrus performed "Like a Prayer" as part of a "Madonna medley" that also included "Express Yourself", and "Music" (2000).

According to Screen Rant, "Like a Prayer" is among the singer's songs that are most used in movies and television. It's been featured in films like Gummo (1997) and Never Been Kissed (1999), and television series like In Living Color (1990), Absolutely Fabulous (1992), and Derry Girls (2018). On April 22, 2024, "Like a Prayer" was used on the trailer for Deadpool & Wolverine. April 22 marked the 35th anniversary of when the song reached the first spot of the Billboard Hot 100; actor Ryan Reynolds reacted to this fact on an X post, writing: "I swear to Marvel Jesus, we did not plan this!".