The CYP3A4 enzyme is known for its atypical inhibition kinetics; ligand inhibition can differ depending upon the probe drug used. A high throughput-LCMS/MS CYP3A4 inhibition assay with four substrate drugs was developed to minimize the potential oversight of CYP3A4 inhibition. The assay uses a 96-well format, human liver microsomes, and four CYP3A4 substrate drugs, midazolam, testosterone, nifedipine and terfenadine. After incubation of the individual substrate with human liver microsomes, the reaction is stopped by solid phase extraction and the four probe metabolites produced are pooled and measured by LCMS/MS with multiple-ion-monitoring mode. Using this assay, the IC(50) values of fourteen compounds recognized as substrates/inhibitors of CYP3A4, were measured for the CYP3A4 catalyzed-metabolism of probe drugs. IC(50) values were also obtained for the common set of compounds by the microtiter plate fluorescent assays with cDNA-expressed CYP3A4. Comparison of the results from the two methods suggests that decision making should be cautiously executed to predict drug interaction potential caused by inhibition of CYP3A4 considering the gap between the two assays and various other factors.