The Spain international came on for Thomas Lemar in the 63rd minute at Mestalla but twisted his knee in the closing stages and was later sent for tests.
An MRI scan has revealed he suffered a grade two sprain of the internal collateral ligament of his left knee.
Atletico have not speculated on his recovery time but there are fears he could miss a number of weeks.
Vitolo made 23 appearances for the club last season, despite being unable to play until January due to a ban on Atleti registering new players.
The former Sevilla favourite posted a tweet on Tuesday, which read: "I'll work to come back as soon as possible. Thanks for your messages of support!"