The test ran between Victoria station and Exchange Square through to Market Street at about 02:00 GMT.
A new tram stop at Exchange Square for Metrolink's Second City Crossing is due to open before Christmas.
The crossing would mean extra capacity for trams and will link St Peter's Square with Victoria station. It is expected to be completed by 2017.
Trams will travel along Princess Street, Cross Street and Corporation Street to reach Victoria.
Transport for Greater Manchester (TfGM) said the scheme was an essential part of the Metrolink expansion.
Peter Cushing, TfGM's Metrolink director, said: "We're at the start of an extensive testing and commissioning process to make sure all the physical infrastructure is ready for us to get passenger services in the run up to Christmas."
The first test trams have run on the new section of track for the second crossing through Manchester.