Dembele is likely to miss upcoming league matches against Real Betis, Espanyol and Villarreal while he has just under four weeks to recover in time for the meeting with Atletico on April 6.
The game at the Camp Nou could prove decisive for Barca, who already sit seven points clear of Diego Simeone's side, their closest challengers at the top of the table.
Dembele appears to have suffered a recurrence of the hamstring strain he sustained during the win over Rayo Vallecano last weekend.
"The tests performed have confirmed that Dembele has a muscle injury in his left hamstring," a Barcelona statement read. "The approximate time out will be three to four weeks."
The Frenchman was not deemed fit enough to start against Lyon but he was risked for the last 20 minutes as the visitors threatened a comeback.
Dembele finished off the 5-1 victory with a goal, latching onto Lionel Messi's pass to confirm Barcelona's place in the quarter-finals.