The 24-year-old has made 28 appearances for Xavi's side since arriving on loan from Atletico Madrid last summer, scoring seven goals and registering five assists in the process.
Felix started against Villarreal in La Liga on Saturday, but he was withdrawn in the 58th minute of what proved to be a 5-3 defeat for the reigning Spanish champions.
The Portugal international had been expected to again be in the squad against Osasuna on Wednesday, but he is now facing a period out with an ankle issue.
"In training on Tuesday morning the first team player Joao Felix suffered a sprain of a ligament in his right ankle. He is unavailable for selection and his recovery will dictate his return," read a statement on Barcelona's official website.
According to journalist Toni Juanmarti, the injury is expected to keep him out of action for the next month, which would see him miss at least six matches for Xavi's side.
Barcelona have league games against Osasuna, Alaves, Granada and Celta Vigo before heading to Napoli for the first leg of their last-16 Champions League clash on February 21.
Felix is set to miss all five of those fixtures, in addition to the La Liga affair with Getafe on February 24, but he could be back for the trip to Athletic Bilbao at the start of March.
The Portuguese has scored four goals and registered two assists in 18 La Liga appearances this season, but his performances have dipped in recent weeks after a positive opening to his Barcelona career.
Barcelona have said on a number of occasions this season that they are keen to sign Felix on a permanent basis during this summer's transfer window.
However, a recent report claimed that the club were considering altering their stance due to a number of disappointing performances.
Atletico extended the forward's contract before letting him leave on loan last summer, with his deal at Wanda Metropolitano due to run until June 2029.
Felix has only managed 34 goals and 18 assists in 131 appearances for the Red and Whites since his arrival from Benfica in 2019, and he spent the second half of last season on loan at Chelsea.