The cocktail substrate approach was frequently used to evaluate Cytochrome P450 (CYP) enzymes mediated drug interactions.The method still remains the challenging in potential interactions among the probe substrates, substrate specificity and sensitivity to the enzymes, and the measurement of liquid chromatography mass spectrometry.Here, we optimized the new method of cocktail substrates to decrease the consumption of time and materials and to increase reliability and accuracy for screening of candidate compounds, and also attempted to expand the method from direct CYP inhibition to time-dependent inhibition (TDI) assay.