The shaggy-haired Spain central defender had not played since Jan. 22 before Saturday's 1-1 La Liga draw at Real, when he was substituted as a precaution after feeling a twinge in a thigh muscle.
"In the end, it was confirmed that the (Barca) captain will be available for (coach) Josep Guardiola," the club said on their website.
The cup final, the first time Barca and Real have met in the competition's title match since 1990, is the second of four meetings between the arch-rivals in 18 days.
They are due to clash in the two-legged Champions League semi-final on April 27 at the Bernabeu in Madrid and May 3 at the Nou Camp in the Catalan capital.
Real coach Jose Mourinho was criticised for employing negative tactics in Saturday's La Liga game.
The Portuguese deployed centre back Pepe as a third defensive midfielder alongside Xabi Alonso and Sami Khedira and left playmaker Mesut Ozil out of his starting line-up.
Guardiola told a news conference on Tuesday he would like to see his players create more scoring chances and said they would remain true to their stylish brand of possession football, whatever tactics Mourinho chose.
"We might change some small details, but the essence won't change," the former Barca and Spain midfielder said.
"Barcelona have virtues that Madrid do not, but Madrid have others that Barcelona do not," he added.
"Whoever imposes their game will win. We will be doing what we can by playing football. We will try to be brave and daring and go out there to win."
Guardiola has been using Jose Manuel Pinto in the domestic cup instead of his first-choice goalkeeper Victor Valdes and he said he would stick with the reserve for Wednesday's game at the Mestalla.
"Tomorrow is about Pinto and 10 more," he said. "It would be a lack of respect to him and the other players if he didn't play."
Full-back Adriano, who complained of a muscle problem after the Real game, was also included in the squad for the trip to Valencia, Barca said.
