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Antibody-drug conjugates (ADCs) consist of monoclonal antibody and cytotoxic drugs covalently attached via stable crosslinkers, and are prospective antibody drugs for cancer therapy. To cover the overall pharmacokinetic understanding of ADCs, both the antibody and the released drugs are necessary for practical clinical observation. The nano-surface and molecular-orientation limited (nSMOL) proteolysis is a universal approach for antibody bioanalysis that enable Fab-selective proteolysis, which maintains antibody sequence specificity while decreasing excess analyte peptides. In this study, we describe quantitative assays for ADC in human plasma using nSMOL for the antibody and polarity-selective liquid–liquid partition with a methanol/ethyl acetate mixed solvent for the cytotoxic drugs. This approach led to the successful development of LC–MS validated bioanalysis of the antibody and released drugs within 20% for lower limit of quantitation and 15% for another concentration setting of Trastuzumab emtansine (T-DM1), Trastuzumab antibody and emtansine conjugated with crosslinker (DM1-MCC). The validated concentration ranges in human plasma were 0.06–250 μg/mL for T-DM1 and 0.39–200 ng/mL for DM1-MCC. These results indicate that LC–MS method with a two-sided approach, using nSMOL and liquid–liquid partition, show potential for the precise pharmacokinetic study for ADC development and treatment.