Bagnaia, on a factory Ducati, arrived in Mugello for this sixth round of the season nursing an ankle injury suffered in a last time out crash in France.
Despite walking with the aid of a crutch once on the bike he proved he is the man to beat in both Saturday's sprint and Sunday's main event.
His quickest lap in the afternoon session was six hundredths of a second faster than Marco Bezzecchi (Ducati-VR46), who is just one point behind Bagnaia in the overall standings.
The Honda LCR team's Spanish rider Alex Rins was placed third ahead of the KTM of South African Brad Binder.
Marc Marquez hit the deck but the six-time champion rushed back to his factory Honda garage to jump on a replacement bike and post the eighth best time.
There was disappointment for 2021 world champion Fabio Quartararo who could only muster the 16th best time on his Yamaha and must finish in the top two in the first qualifying session on Saturday to go through to the second qualifying session and a shot at pole.
