The Italian will be without centre-back pairing Raphael Varane and Pepe for the trip to Mestalla, the former is absent with a knee injury while the latter is suspended after collecting his fifth booking of the season in the 2-2 draw at Osasuna.
Ancelotti did receive a boost in midweek, however, as Sergio Ramos' red card that he collected in the Osasuna fixture was rescinded.
Nacho was chosen to feature in Real's 2-0 victory over Xativa in the Copa del Rey on Wednesday, and the 23-year-old could fill the void alongside Ramos.
Real are looking for a swift response to the disappointing draw at Osasuna, where second-half strikes from Isco and Pepe not cancelled out Oriol Riera's double.
The draw ended a run of five straight victories for the Liga giants, and saw the gap to leaders Barcelona and second-placed Atletico Madrid extend to five points.
Recent history suggests Real will emerge victorious, with the Bernabeu outfit registering victories on their last four visits to Mestalla in the league.
Indeed, Real have not lost in their previous 10 encounters with Valencia in all competitions.
But Ancelotti will be wary of a Valencia side desperate to make a point after head coach Miroslav Djukic was relieved of his duties last week.
The 47-year-old was only handed the reins in June but paid the price for an inconsistent campaign that has seen Valencia slip to ninth in the standings.
A 3-0 setback to high-flyers Atletico proved to be his last game in charge, with youth coach Nico Estevez handed temporary charge of the side.
His interim tenure began with a 1-0 triumph over Gimnastic in the Copa del Rey with Paco Alcacer settling the tie.
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