Barcelona confirmed that the young Spaniard would miss the game due to a muscle injury. There is no timeline on when he will be returning to the pitch.
"Pedri is out with a left thigh injury and his recovery will dictate his return," Barcelona said in a statement.
The news comes as a major blow to Ronald Koeman, whose side have a tricky run of fixtures coming up. Following their game against defending La Liga champions Atletico Madrid, Barcelona face Valencia and Real Madrid after the international break.
However, in some positive news, Jordi Alba is set to make his return to the side. The left-back missed the last few games with a muscle injury but is available for the trip to Atletico.
Pedri was passed fit hours before Barcelona faced Benfica
Pedri has had a physically and emotionally exhausting few months. The Spaniard was a key part of Barcelona's side last season and then went on to represent Spain in Euro 2020 and the Tokyo Olympics.
The 18-year-old barely had any time off and was called into action for Barcelona just days after he returned from Japan. The lack of rest has taken its toll on Pedri, who has not looked in great shape this season.
Last month, Barcelona confirmed Pedri had picked up a thigh injury.
"The first team player Pedri has a quadriceps muscle injury in his left thigh. The player is unavailable for selection and his recovery will dictate his return. Pedri was in the starting XI against Bayern Munich on Tuesday and played all 90 minutes. In total this season the midfielder has made two appearances in the league - the 4-2 win over Real Sociedad at Camp Nou and the 1-1 draw away at Athletic Club in San Mamés."
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Pedri was given the green signal to play against Benfica in the Champions League, but that decision appears to have backfired. The Spaniard played over an hour against the Portguese giants, but has come off worse for wear.