The league leaders are four points clear with two games to play after earlier claiming a comfortable 2-0 win over David Moyes's Real Sociedad thanks to goals from Neymar and Pedro Rodriguez yesterday.
Barcelona can now seal the title when they travel to reigning champions Atletico Madrid next Sunday should a potential players' strike due for the weekend be called off.
First-half goals from Paco Alcacer and Javi Fuego gave Valencia a commanding half-time lead.
"There are two games to go, we need to win them, but obviously it is more difficult (to win the league)," admitted Madrid boss Carlo Ancelotti.
However, the Italian refuted suggestions the result could be a blow to his side's confidence ahead of their crucial Champions League semi-final, second-leg against Juventus on Wednesday.
"The result isn't good, but the performance was the complete opposite. We have a lot of chances, hit the post three times, missed a penalty, sometimes it just isn't enough.
Alcacer wasted a big early chance when he spooned over when clean through on Iker Casillas, while at the other end Gareth Bale saw a brilliant free-kick come back off the bar.
Ronaldo then also sent a header come off the frame of the goal, but it was Valencia who opened the scoring when Madrid-target Jose Gaya's excellent low cross was turned home by Alcacer.
Madrid suffered a huge injury blow ahead of the Juventus clash as Toni Kroos limped off to be replaced by Asier Illarramendi.
Alves then made an incredible save from a Bale piledriver before the Welshman teed up Javier Hernandez to hit the woodwork for the third time in the half.
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