Top scorer in both La Liga and the Champions League this season, Ronaldo has sat out Real's last two matches after developing knee and thigh muscle problems in his left leg.
Ancelotti confirmed the 29-year-old would not be available for Saturday's La Liga game at home to Almeria but said the fact Real retained some hope he might feature against Barca showed the injuries were not serious.
"I don't know if he will be able to play on Wednesday," Ancelotti told a post-training news conference.
"He is not happy because it is an important moment in the season," added the Italian.
"If he is not at 100 percent he won't play. If he has a chance of featuring in the Cup final that means it's not a serious injury."
Losing Ronaldo for an extended period would be a blow to Real's hopes of claiming a rare treble of titles.
They are third in La Liga, three points behind leaders Atletico Madrid and two adrift of Barca, and play Bayern Munich in their Champions League semi-final, first leg on April 23.
Barca became the only Spanish team to win Europe's elite club competition, La Liga and the King's Cup in the same season under Pep Guardiola in 2008-09.
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