The pair missed Saturday's 2-1 La Liga victory at Deportivo La Coruna, when Mourinho initially left top-scorer Cristiano Ronaldo on the bench but brought him on in the second half to set up Gonzalo Higuain's late winner.
Asked if Spain international Alonso would be fit for the "Clasico" at the Nou Camp, a typically brusque Mourinho told a news conference: "I think so".
Quizzed a few minutes later on the fitness of France's Benzema, he added: "He trained with the squad yesterday and today and it seemed to me he is in the right physical condition to play."
Real, who host runaway leaders Barca in La Liga on Saturday, are on the back foot in the cup after Barca secured a 1-1 draw at the Bernabeu in last month's first leg.
Cesc Fabregas put the visitors ahead five minutes after halftime before Raphael Varane nodded an 81st-minute equaliser.
Tuesday's match will be the 224th meeting between the great rivals in all competitions. Barca have won 87 to Real's 88 and there have been 48 draws.
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