From Staunton east to Richmond, the highway serves as the local complement to Interstate 64 (I-64), roughly following the 18th century Three Notch'd Road through Waynesboro and Charlottesville on its way through the Shenandoah Valley, its crossing of the Blue Ridge Mountains at Rockfish Gap, and the Piedmont.

[1][2] US 250 passes to the north of Jack Mountain and follows Crab Creek through a gap in Doe Hill to McDowell, where the highway crosses the Bullpasture River.

After passing through Shaws Ridge, US 250 goes through the hamlet of Head Waters and has a curvaceous crossing of Shenandoah Mountain, where the highway enters Augusta County.

At the Augusta County border, a parking area leads to Fort Edward Johnson, which was a series of fortifications built by the Confederate Army in 1862.

The highway meets SR 262 (Woodrow Wilson Parkway) at a diamond interchange at the west city limits and heads southeast toward the downtown area.

SR 254 heads north on Coalter Street and east with northbound US 11 on Commerce Road while US 250 and US 11 pass under the railroad together on Greenville Avenue.

[1][2] US 250 heads east as a four-lane undivided highway through the southeastern part of Staunton, passing the remains of Western State Hospital and intersecting SR 261 (Statler Boulevard).

The two highways pass under Norfolk Southern Railway's Roanoke District and diverge at Delphine Street, where US 340 heads north toward Elkton.

US 250 veers away from I-64, passes under the CSX rail line, and has a junction with the northern end of SR 151 (Critzers Shop Road) at the bottom of the descent.

The highway parallels the railroad to just west of Charlottesville, where US 250 meets US 29 (Monacan Trail Road) at a partial cloverleaf interchange.

US 250 Business continues east along Ivy Road through the University of Virginia campus while US 250 joins US 29 northward on the freeway bypass.

[1][2] US 250 meets the eastern end of US 250 Business and SR 20 (High Street) just before crossing the Rivanna River on the Free Bridge to re-enter Albemarle County.

US 33 and US 250 intersect SR 161 (Boulevard) northwest of the Science Museum of Virginia, in the former Broad Street railroad station.

US 250 Business begins at the partial cloverleaf interchange where US 250 joins US 29 (Monacan Trail Road) on its bypass of Charlottesville.

US 250 Business passes to the south of the University of Virginia sports complex, which includes Davenport Field, Klockner Stadium, and John Paul Jones Arena.

[1][4] At The Corner, US 250 Business leaves the university grounds and veers onto Main Street, which passes under the CSX rail line.

The business route passes Charlottesville Union Station at its crossing of Norfolk Southern Railway's Washington District.