The eastern terminus is at U.S. Route 30 (US 30) in East Whiteland Township, a short distance west of Malvern.

PA 401 is called Conestoga Road its entire length and runs through rural areas with suburban development toward the eastern part of the route.

After this junction, the road runs between housing developments to the north and rural areas to the south, crossing into West Nantmeal Township.

The road curves south before continuing southeast into West Pikeland Township, where it crosses Pickering Creek before intersecting PA 113.

The route runs past more housing developments and crosses Valley Creek before it widens to four lanes at the Phoenixville Pike intersection and reaches a diamond interchange with the US 202 freeway, where it is briefly a divided highway.

The route narrows back to a two-lane undivided road past this interchange and crosses the Chester Valley Trail before continuing southeast to the eastern terminus at US 30 (Lancaster Avenue) in Frazer.