Francesco Bagnaia je postavil najhitrejši čas (1:44.938) drugega prostega treninga v Mugellu. Presenetljivo drugi je bil tokrat Alex Rins, tretji pa Pedro Acosta.
Miguel Oliveira je kot najboljši dirkač na Aprilinem motociklu postavil četrti čas, pred štirimi Ducatijevemi dirkači Marcom Marquezom, Eneo Bastianinijem, Jorgejem Martinom ter Alexom Marquezom.
Z devetim in desetim mestom sta si neposreden nastop v hitrejši kvalifikacijski skupini uspela zagotoviti še Maverick Vinales ter Aleix Espargaro.
Rezultati:
| Poz | Dirkač | Drž | Moštvo | Čas/Zaostanek | Krogi | Max hitrost |
| 1 | Francesco Bagnaia | ITA | Ducati Lenovo (GP24) | 1’44.938s | 18/20 | 358k |
| 2 | Alex Rins | SPA | Monster Yamaha (YZR-M1) | +0.273s | 22/23 | 359k |
| 3 | Pedro Acosta | SPA | Red Bull GASGAS Tech3 (RC16)* | +0.388s | 17/18 | 362k |
| 4 | Miguel Oliveira | POR | Trackhouse Aprilia (RS-GP24) | +0.402s | 20/22 | 359k |
| 5 | Marc Marquez | SPA | Gresini Ducati (GP23) | +0.407s | 22/24 | 358k |
| 6 | Enea Bastianini | ITA | Ducati Lenovo (GP24) | +0.465s | 22/22 | 352k |
| 7 | Jorge Martin | SPA | Pramac Ducati (GP24) | +0.467s | 23/25 | 359k |
| 8 | Alex Marquez | SPA | Gresini Ducati (GP23) | +0.474s | 22/24 | 356k |
| 9 | Maverick Viñales | SPA | Aprilia Racing (RS-GP24) | +0.486s | 18/23 | 355k |
| 10 | Aleix Espargaro | SPA | Aprilia Racing (RS-GP24) | +0.523s | 16/18 | 359k |
| 11 | Fabio Quartararo | FRA | Monster Yamaha (YZR-M1) | +0.542s | 19/21 | 360k |
| 12 | Fabio Di Giannantonio | ITA | VR46 Ducati (GP23) | +0.712s | 23/23 | 359k |
| 13 | Brad Binder | RSA | Red Bull KTM (RC16) | +0.760s | 22/23 | 361k |
| 14 | Marco Bezzecchi | ITA | VR46 Ducati (GP23) | +0.791s | 26/26 | 361k |
| 15 | Franco Morbidelli | ITA | Pramac Ducati (GP24) | +0.821s | 21/22 | 354k |
| 16 | Raul Fernandez | SPA | Trackhouse Aprilia (RS-GP23) | +0.846s | 16/21 | 354k |
| 17 | Jack Miller | AUS | Red Bull KTM (RC16) | +0.860s | 16/21 | 360k |
| 18 | Pol Espargaro | SPA | KTM Test Rider (RC16) | +1.140s | 17/19 | 366k |
| 19 | Takaaki Nakagami | JPN | LCR Honda (RC213V) | +1.141s | 21/22 | 359k |
| 20 | Johann Zarco | FRA | LCR Honda (RC213V) | +1.576s | 21/23 | 360k |
| 21 | Joan Mir | SPA | Repsol Honda (RC213V) | +1.646s | 17/21 | 359k |
| 22 | Lorenzo Savadori | ITA | Aprilia Racing (RS-GP24) | +1.802s | 15/21 | 352k |
| 23 | Augusto Fernandez | SPA | Red Bull GASGAS Tech3 (RC16) | +1.936s | 19/21 | 359k |
| 24 | Luca Marini | ITA | Repsol Honda (RC213V) | +2.124s | 22/22 | 358k |
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