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Indiana to Cincinnati
US 50 enters Ohio from Indiana at a traffic light with State Line Road. The route heads northeast as a four-lane undivided highway, passing through farmland and paralleling a railroad track. The highway passes under Interstate 275 before entering the extreme western suburbs of the greater Cincinnati area. The road has an at-grade crossing with a railroad track, before curving southeast. US 50 has a traffic signal at the intersection with SR 128. Southeast of SR 128, US 50 becomes a four-lane divided highway and cross over the Great Miami River. After the river the route enters Cleves and curves due south and passes through residential properties. In Cleves the highway has a traffic signal with SR 264, before leaving Cleves and entering North Bend. In North Bend the road continues to pass through woodland and residential properties, before passing the monument and tomb of President William Henry Harrison. The highway curves east-southeast and becomes a four-lane undivided highway, before leaving North Bend. The road parallels the Ohio River and passes through a mix of woodland, residential and industrial properties. The route begins to curve northeast and enters Cincinnati.

Cincinnati
In Cincinnati it is routed along the Sixth Street Expressway, Fort Washington Way, and the historic Columbia Parkway (from which trucks are banned).

Sixth Street Expressway
The Sixth Street Expressway is a 1.19 mi six-lane urban controlled-access highway that runs from Interstate 75 in Queensgate, just west of downtown Cincinnati, Ohio, to the Waldvogel Viaduct, which continues to Lower Price Hill. The expressway opened to traffic in 1964, replacing Sixth Street for most of its length.

Fort Washington Way
Fort Washington Way is a one-mile section of freeway in downtown Cincinnati, Ohio. Running east-west in a trench, it carries Interstate 71 and US-50, first built between 1958 and 1961. It is named for a former fort which once stood nearby.

Its western terminus is with Interstate 75. At this location, Interstate 71 is multiplexed with Interstate 75 and turns south, crossing the Ohio River on the Brent Spence Bridge. US-50 continues westbound as the Sixth Street Expressway and then as River Road.

Fort Washington Way's eastern terminus is at the Lytle Tunnel, where Interstate 71 turns north. US-50 continues east across the Third Street Viaduct onto Columbia Parkway.

Much of Cincinnati's historic riverfront below Third Street, was cleared in the late 1950s for the construction of Fort Washington Way.

Columbia Parkway
Columbia Parkway is an expressway/parkway extending from Downtown Cincinnati, Ohio to the Village of Fairfax, and serving as US 50 on Cincinnati's east side. It was constructed in the Art Deco architectural style in 1938 as part of the New Deal. The Parkway's western terminus is Fifth Street, near Fort Washington Way (which carries Interstate 71 and US 50 through downtown Cincinnati).

The road has a "Dead Man's Curve," between Tusculum Avenue and Beechmont Avenue, which is often the site of accidents.

Columbia Parkway is a partially limited access roadway, with crude on and off ramps to many local streets as well as conventional intersections. Westbound Columbia Parkway provides access to Red Bank Road, Eastern Avenue, Tusculum Avenue, Stanley Avenue, Hoge Street, Delta Avenue, Torrence Parkway, William Howard Taft Road, Kemper Lane, Eggleston Avenue, Fort Washington Way/Interstate 71, Sixth Street, Interstate 471, and Fourth Street/Pike Street. Eastbound Columbia Parkway provides access to Riverside Drive/Bains Street, Kemper Lane, William Howard Taft Road (access restored October 2008), Torrence Parkway, Delta Avenue, Hoge Street, Stanley Avenue, Tusculum Avenue, Beechmont Avenue, and Red Bank Road.

The section of the roadway between William Howard Taft Road/ Torrence Parkway and Delta Avenue has numerous abandoned staircases built into the Art Deco retaining walls (which were constructed in 1938 as part of the Works Progress Administration). The City of Cincinnati began the process of sealing up these staircases in 2008.

Cincinnati to West Virginia
In Fairfax, US 50 becomes known as Wooster Pike and crosses the Little Miami River via the Wooster Pike Bridge in Milford. East of Milford, US 50 becomes a two-lane highway as it travels through Hillsboro and Bainbridge before reaching the outskirts of Chillicothe. In Chillicothe, US 50 becomes Western Avenue and then Main Street before merging with U.S. Route 23 and U.S. Route 35 east of the city. State Route 104 overlaps US 50 through downtown Chillicothe. As US 50 leaves Chillicothe it becomes a two-lane highway once again as it winds its way through McArthur and heavy forested Southern Ohio before reaching the outskirts of Athens where it becomes a four-lane highway. It will remain four-lane until reaching the Ohio River. US 50 joins with State Route 32 near the Ohio University Bush Airport–Snyder Field in Albany, west of Athens. US 50 intersects with U.S. Route 33 east of the city before continuing eastward toward Belpre and the Ohio River. US 50 begins to overlap SR 7 near Reedsville as it heads north along the river, and splits from SR 7 before it crosses the river on the Blennerhassett Island Bridge to Parkersburg, West Virginia.