The Italian coach insists his side will not be affected by its recent 4-3 defeat to the Blaugrana and feels a cup victory will spur it on as it looks to secure the treble.
The Spanish giants meet in the cup final Wednesday at the Mestalla to battle it out for their first piece of silverware of the season.
Both sides are currently challenging for La Liga as they trail current leaders Atletico Madrid, while Ancelotti's men are also in the final four of the Champions League.
The Italian coach believes lifting the Copa del Rey at the expense of the club's fierce rivals will give his side a massive mental boost in order to maintain its fight to win the treble this season.
"This game is important for a lot of reasons," Ancelotti said on his club's official website. "Firstly, it is the first title of the season that we can win. Secondly, we will take on a great side like Barcelona. All of this gives the game a lot of importance. The possibility of winning the first trophy of the season is motivation for us.
"On a mental level, I think it will be very important. I think after this game, the side that wins will have more motivation, illusion and desire to play their best until the end of the season. I think from a mental point of view, it will be very important."
The former AC Milan boss added that he feels his side's 4-3 defeat to Gerardo Martino's men in March will have no effect on his players as they head into Wednesday's clash.
"I don’t think that there will be a psychological advantage for what happened in the past. The present is a complicated game for both Barcelona and Real Madrid. We have to play well, with excitement, motivation and with a lot of concentration to try and win it," he said. "In January we played very well. In the cup we didn’t concede a goal, we played well…I think, above all, that we deserve to be in the final and I’d tell the fans to go into the game with excitement, with the same desire that the team has, that we are all going to give our all to win this title."
The coach, who has coached in Italy, England, France and Spain, was asked about the differences in styles between the four countries.
"In Italy tactics are very important," he replied. "In England there is a rapid and intense rhythm, in France a lot of intensity and physical force, and in Spain it is about taking care of the ball.
"There are two players who play in my area and could be likened to me: Xabi Alonso and Asier Illarramendi."