About 2 hours by train, one easy change. The Basilica of San Francesco with Giotto's frescoes is worth the trip alone. But the whole town rewards a full day.
No direct train. Best: Florence → Terontola → Assisi (2h10) or Florence → Foligno → Assisi (2h30). Station is Assisi-Santa Maria degli Angeli in the valley — take Line C bus up (8 min, €1.30).
A1 south to Valdichiana, then E45 to Assisi. Park at Parcheggio Giovanni Paolo II (Piazza Matteotti) — €1.65/h.
Train with one change. Leave Florence by 8am, arrive by 10am, full day in the town. Bring good shoes — all hills.