The club announced that the World Cup-winning centre-back sustained a problem in the final training session before the players were due to take a flight east.
Sergi Roberto is also absent for the Catalan side, while Carles Alena and Carles Perez have been overlooked by head coach Ernesto Valverde.
In a statement posted on the club website, the Camp Nou side reports: “In the final part of the session on Tuesday at the Ciutat Esportiva, Samuel Umtiti picked up a bruised knee and so the French central defender misses the trip to take on Slavia Prague.
The 25-year-old has struggled to make an impression on the starting XI this season with Barca, featuring only once – in the weekend’s 3-0 win over Eibar.
He has been plagued by injury problems, with a foot issue pinning him to the sidelines for a month and preventing him from featuring in five matches.
Gerard Pique, who was suspended at the weekend after picking up five yellow cards in the first eight matches of the Primera Division season, will step back into the team to play alongside Clement Lenglet.
Having struggled in the early part of the season, winning just two of their opening six fixtures, the defending Primera Division champions appear to have found their feet once again, winning five matches in succession.
Among those was a thrilling come-from-behind success over Inter in their previous Champions League outing, carrying them to a healthy four points from the opening two rounds. Previously, they had drawn scoreless away to Borussia Dortmund.
They will expect to keep this positive run going against Slavia Prague, who are expected to be the whipping boys in Group F, despite coming within a whisker of upsetting Inter on the opening night of action. Subsequently, the Czechs lost 2-0 at home to BVB.Barcelona squad to face Slavia Prague: Ter Stegen, N. Semedo, Pique, I. Rakitic, Sergio, Todibo, Arthur, Suárez, Messi, O. Dembele, Neto, Lenglet, M. Wague, Griezmann, Jordi Alba, F. De Jong, Vidal, Junior, Inaki Peña, Ansu Fati.