The Spain international midfielder suffered a deep cut to his right knee after colliding with Real Sociedad defender Carlos Martinez during the closing stages of Saturday's 2-1 victory over the Basques.
Guardiola admitted during his pre-game press conference on Tuesday evening that Busquets was his "most doubtful" injury concern for the visit of Los Che.
However, the Catalan coach added: "Xavi's fine, Alexis [Sanchez] is doing pretty well and we will see tomorrow how Andres [Iniesta] and Pedro are."
Turning his attention to the opposition, Guardiola said that he is expecting Valencia to push Barca just as hard as Real Madrid did in the quarter-finals.
With the tie delicately poised at 1-1 after last week's first-leg clash at Mestalla, the 41-year-old is predicting things will go right down to the wire.
"Valencia have always maintained a very high level and the opinion I have regarding them remains the same," he explained.
"As against Madrid, I have a feeling that the tie will not be decided until the last minute."
Jonas opened the scoring for Valencia at Mestalla last week only for Barca to equalise through Carles Puyol before Lionel Messi missed a second-half penalty for the visitors.
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