Reigning champion Bagnaia hit a fastest lap time of one minute and 30.685 seconds at his home 'Marco Simoncelli' circuit in Misano Adriatico, 0.185sec ahead of six-time champion and title challenger Marc Marquez.
Ducati rider Bagnaia is competing through shoulder, neck and collarbone pain after crashing out of last weekend's Aragon MotoGP, an accident which left him 23 points behind current championship leader Jorge Martin.
Martin, who rides for Ducati satellite team Pramac, was fastest in Friday morning's free practice and finished the afternoon session 0.281sec off the pace in third.
Martin has not won a Grand Prix since the France back in May but won at Misano last year. The 26-year-old has also finished second in both the sprint and main race in four of the last five GPs.
Both Bagnaia and Martin were comfortably in the top 10 positions which gives riders direct access to the second round of qualification on Saturday morning.
Qualifying shapes the grid for the sprint on Saturday afternoon and the main event GP on Sunday, with a maximum of 37 points available.