Ketoconazole is an azole antifungal drug used for the treatment of fungal infections. However, ketoconazole is not widely used due to its liver toxicity. Ketoconazole is a chiral drug and is currently administered as the racemic mixture. Recent studies have shown that the enantiomers have different in vitro activity; therefore, the stereochemistry of ketoconazole could modulate its biological effects. Previous HPLC assays quantified ketoconazole as the racemate and limits of quantitation >12 ng/mL were achieved. While some non-bioanalytical assays were able to resolve the two ketoconazole enantiomers, these assays had long HPLC run times (>30 minutes). Here we report on an accurate and precise validated LC/MS/MS assay for the determination of the enantiomers of ketoconazole from human and dog plasma.