The Portuguese has missed his side's last few matches with a hamstring injury, which is expected to keep him on the sidelines for the next 10 days.
Ancelotti hinted on Monday that his leading scorer could yet make an appearance, but the Italian has today confirmed that the attacker will play no part in Valencia.
"I found out that I can't count on Cristiano this morning. We can do well tomorrow despite the absence of some players," Ancelotti told reporters at a press conference.
Ronaldo has scored 45 times in all competitions this season.