Captain Iniesta did not train with the rest of the squad on Wednesday (AEST) and has been omitted from the 19-man party named for the trip to the Santiago Bernabeu.
The midfielder, whose issue is with his right leg, joins Rafinha (adductor) and Thomas Vermaelen (calf) in missing out through injury.
Iniesta played 68 minutes of the first leg as Barca fell to a 3-1 defeat, leaving them with a mountain to climb when it travels to the capital on Thursday.
A Gerard Pique own goal gave Madrid the lead before Cristiano Ronaldo and Marco Asensio clinched the victory with Lionel Messi having pulled Barca level from the spot.
Madrid will be without Ronaldo for the return leg after the Portuguese was banned for five matches after a shove on referee Ricardo de Burgos Bengoetxea in Barcelona.