A common approach to characterize ADC biotransformation utilizes hybrid immunoaffinity capture coupled with intact protein mass LC-high resolution MS (LC-HRMS). First, the ADC is selectively enriched from samples through immunocapture, then eluted off the substrate before being analyzed through LC-HRMS. Finally, data analysis and structure elucidation rely on protein deconvolution by integrating information from RT -m/z domain to RT -deconvoluted mass domain. Through comparing the changes in deconvoluted mass, scientists may identify and propose the potential structure of biotransformation species. Previously, our group employed this method to understand the biotransformation profiles of multiple ADCs with different designs and therapeutic purposes. Especially, for AZD8205, an B7H4-directed ADC, with drug to antibody ratio (DAR) equal to 8, we observed several important biotransformation phenomena, including the progression of thio-succinimide linker hydrolysis and linker-payload deconjugation in vivo. Interestingly, we also found a product that represents the disulfide bond reformation between antibody light chain (Lc) and heavy chain (Hc), as a cysteine-conjugated DAR 8 ADC should not contain inter-chain disulfide bonds. Although the intact protein mass LC-HRMS method is robust and routinely used for characterization of ADC biotransformation, the structure information still heavily relies on the deconvoluted mass changes. Further structural confirmation and characterization improves confidence in the findings reported. This work aimed to develop a method that is suitable for in-depth structural elucidation of ADC biotransformation species. Immunoaffinity capture allows selective isolation of ADCs from matrices, while on-beads tryptic digestion yields peptides containing possible biotransformation modifications ready for assessment by LC-MRM. The bottom-up method avoids the possible limitation where biotransformation peaks are overwhelmed by parent/untransformed ADC peak in intact protein mass LC-HRMS. Furthermore, MS/MS fragmentation enabled detailed structural confirmation of biotransformation products through mass fingerprinting. Many of the findings in this work support our earlier findings. Collision induced dissociation (CID) fragmentation informed specific localization of linker-payload deconjugation, whereas intact mass LC-HRMS is unable to discern site-specific changes. We also highlight the advantages of newly developed electron activated dissociation (EAD) fragmentation. Previously, scientists employed EAD to investigate the glucuronidation and oxidation metabolism of a small molecule therapeutic. In our case, EAD not only aided in distinguishing the isomerism of thiosuccinimide hydrolysis, but also provided supplemental information on disulfide bond-linked biotransformation species, due to its preferential fragmentation of disulfide bonds. Herein, we present the first such detailed structural confirmation of biotransformation products of an ADC from in vitro samples using a LC-MRM approach. The findings support the prior conclusion that AZD8205 is structurally stable, with very minor fraction going through unique biotransformation reactions requiring exquisite instrumentation and advanced analytical workflows for detection and characterization.