The Spain international picked up a hamstring injury during the first leg of the clash with the Blaugrana and was left off the bench for Diego Simeone's side.
The Spain international, who leads his side in scoring this season, limped off midway through the first half of the first leg at Camp Nou last week.
The capital club confirmed that he did not suffer a hamstring tear and had been hopeful that he could feature during the Vicente Calderon on Wedensday.
However, he has not recovered sufficiently to line up alongside his teammates or even take a place on the bench. Instead, Adrian Lopez stars up front alongside David Villa.
ATLETICO MADRID: Courtois; Juanfran, Miranda, Godin, Filipe; Gabi, Koke, Tiago, Raul Garcia; Adrian, Villa
BARCELONA: Pinto; Alves, Mascherano, Bartra, Alba; Busquets, Xavi, Iniesta; Messi, Fabregas, Neymar