The Aruba.it rider, who regularly rides at the Misano World Circuit in his test role for the manufacturer, set a 1m31.909s to top the timesheet on Friday morning.
He led from Marini, just one-tenth slower, who was confirmed on Thursday to be staying with VR46 for 2024, with Pramac’s Jorge Martin in third.
Championship leader Francesco Bagnaia, who escaped serious injury in a crash at last weekend’s Catalan GP, looked sore as he put in an early stint on his factory Ducati.
The reigning world champion was left with severe bruising after a massive highside in Sunday’s race before he was hit by Brad Binder’s KTM.
After an initial six-lap run, he took a break before going back out and ended the session in 20th with a 1m32.599s.
Fabio Di Giannantonio set an early benchmark on his Gresini Ducati, leading with a 1m35.889s, before Martin went quicker by 0.3s.
Pirro put in the first sub 1m35s time soon after, with a 1m34.507s, before a bruised Marco Bezzecchi, who was caught up in last weekend’s Turn 1 crash at Barcelona, went quickest with a 1m33.987s.
The VR46 rider was wiped out by Enea Bastianini, who sits out this weekend’s round, and suffered an injured left hand for which he is receiving thrice-daily physiotherapy.
The Aprilia pair, who claimed an historic 1-2 for the Noale factory last Sunday, then went fastest, with winner Aleix Espargaro ahead of Maverick Vinales.
Pirro soon reclaimed the top spot ahead of Bezzecchi, while fellow wildcard Dani Pedrosa, riding for KTM, slotted into second as he tested a carbon fibre chassis.
With 20 minutes remaining, Pedrosa went quickest with a 1m32.250s, leading from Pirro and Martin, who was 0.2s off the pace.
Marini then shot up to second, having been 10th, before he improved again to run 0.1s behind leader Martin, with a 1m32.066s.
Pirro then went fastest as Marini slotted into second to end the session ahead of Martin.
Bezzecchi was fourth ahead of RNF Aprilia’s Raul Fernandez, who put in a 1m32.227s in the closing stages.
Pedrosa was sixth ahead of Vinales, Zarco, Aleix Espargaro and Gresini’s Alex Marquez completed the top 10.
Franco Morbidelli was the fastest of the struggling Japanese bikes in 12th, with team-mate Fabio Quartararo in 15th.
Marc Marquez finished in 21st for Honda as rumours continue to swirl about his future at the team.
Augusto Fernandez was the only fall of the session, sliding off his Tech3 GasGas KTM at Turn 14 early on.
