Carlo Ancelotti has ruled the Welshman out of Wednesday's game with Olimpic de Xativa, with Angel Di Maria taking his place as Real Madrid aims to reach the round of 16.
The Santiago Bernabeu side was held to a scoreless draw in the first leg of the round of 32 tie and will be without the Wales international when the club attempts to book its ticket for the final 16 later this week.
Bale picked up a minor knock in Tuesday's training session and will be replaced by Angel Di Maria for the encounter in the capital.
"Gareth has picked up a small injury during training and will not be available for Wednesday's game," Ancelotti said at a press conference. "Di Maria will take his place in the starting XI. Angel didn't play well against Osasuna, but he will get another chance on Wednesday.
"We will have to wait and see how Bale's injury develops, but I do not expect any problems for Sunday's game against Valencia."
Ancelotti then went on to discuss Madrid's title credentials in La Liga and claimed the club will have to get close to a record points tally in order to reclaim the title.
"We all know that we will have to get around 100 points in order to win La Liga," added the Italian.
Madrid currently sits third in the league table with 38 points from 16 games, trailing joint leaders Barcelona and Atletico Madrid by five points.