Automated high throughput sample analysis by HTLC-MS/MS has increasingly become the method of choice for pharmaceutical/biopharmaceutical industries in virtually all phases of drug discovery and development. HTLC-MS/MS was utilized for in-vitro predictive ADME including multi-drug (protein/peptide drugs) analyses of inhibition studies using a variety of P450 isozymes. A generic program was used to support high throughput drug discovery applications such as multi-drug protein binding study to compare with equilibrium dialysis techniques. Multi-drug (protein and peptide molecules) analyses were performed using the Cyclone® extraction columns (normal and micro sizes) regarding extraction efficiency/recovery and matrix effect. Method precision, accuracy, throughput and ruggedness were also evaluated with acceptable results for quantitative analyses. This automated technique has shown great advantages in the effectiveness and ruggedness of analyzing multi-drugs and their metabolites simultaneously over traditional HPLC and off-line LC-MS/MS techniques.