The Catalans will be desperate to avoid a third straight defeat in the Copa del Rey final after being knocked out of the Champions League and losing to Granada in La Liga
The two giants of Spanish football will be keen to seize on the opportunity to land a psychological advantage ahead of the Liga title run-in, with both currently trailing surprise leaders Atletico Madrid.
Barca and Real have already met twice in the league this term - Barca coming out on top both times - and their most recent meeting was one of the most explosive Clasico clashes in recent years.
With seven goals, three penalties and a red card, March's contest surpassed expectations in terms of drama, but both sides would presumably settle for a more straightforward affair at Mestalla on Wednesday.
Since seeing off Real Madrid at the Santiago Bernabeu last month, Barca have crashed out of the Champions League following their defeat to Atletico, while also slipping to third in the table.
They were beaten 1-0 by Granada on Saturday and face an uphill task to retain their league crown - adding even more importance to this fixture.
Real, meanwhile, were able to shake off the defeat to Barca to move into the semi-finals of the Champions League and they remain on Atletico's heels thanks to dominant wins over Rayo Vallecano, Real Sociedad and Almeria in recent weeks.
In what will be the seventh Copa final between the sides, Ancelotti is able to welcome back Sergio Ramos (neck), although Marcelo (hamstring), like Cristiano Ronaldo, is a doubt.
Barca skipper Puyol may be required to return from a series of knee problems sooner than anticipated due to the injuries being carried by defensive pair Gerard Pique (pelvis) and Marc Bartra (hamstring).