This paper describes a broadly applicable approach for in-depth investigation of ADC biotransformation products from in vitro or in vivo studies. The process includes specific enrichment of ADC from samples through immunoaffinity capture, on-beads tryptic digestion, followed by analysis using LC-MRM-MS. The employment of two orthogonal fragmentation methods, CID and EAD, provided complementary results in the characterization of AZD8205 is an anti-B7H4 Topoisomerase I inhibitor ADC with an average DAR of 8. The linker-payload (PL) contains maleimide ring, polyethylene glycol (PEG), Val-Ala dipeptide cleavable linker and AZ14170132 payload. The linker-payload was conjugated to the anti-B7H4 antibody through reduction of all four interchain disulfide bonds, followed by thiol-maleimide reaction. Trypsin digestion of captured AZD8205 yields three peptides linked with linkerpayload: GEC from antibody light chain (Lc), SCDK from antibody heavy chain (Hc) and THTCPPCPAPELLGGPSVFLFPPKPK (THT peptide) from the hinge region in Hc, as shown in Figure . All four cysteines in the peptide sequences referenced above were conjugated to linker-payload.