From the north (Milan, Florence), the best exit for Assisi on the A1 motorway at Valdichiana, where the superstrada SS75bis branches into Umbria at Lake Trasimene and continues towards Perugia-Assisi.

From Rome travelling northwards along the A1 motorway the best exit for reaching Assisi is at Orte, which is connected to the regional capital at first by the ss204 to Terni and then by the E45 (or SS3bis) from Terni to Perugia-Assisi.
The A14 motorway down the Adriatic coast of Italy has an exit at Cesena for the E45 "superstrada" to Città di Castello and Perugia-Assisi.