In conclusion, we employed tryptic digestion and LC-MRM approaches with two orthogonal fragmentation methods: CID and EAD to confirm and further characterize our previous findings of AZD8205 biotransformation. Overall, bottom-up LC-MS/MS not only enabled the identification of low concentration biotransformation products, but also provided further structural details. By applying CID, we characterized the progression of thio-succinimide hydrolysis over time. The obtained results align with the analyses performed using previously reported intact protein mass LC-HRMS method. This study once again confirmed that the structural stability of AZD8205 originated from the high degree of thio-succinimide linker hydrolysis. Besides, CID aided in the investigation of the site preference of Cys or GSH adduct on Hc. We noticed that the 1 st conjugation site on Hc hinge region showed a slightly higher possibility of Linker-PL deconjugation followed by forming thiol adduct. Additionally, we found disulfide reformation between antibody subunits and the formation of an intra-chain disulfide bond on Hc. We also highlight the application of EAD fragmentation for providing additional details in the characterization of biotransformation products. Unique diagnostic ions generated from EAD supported the differentiation of constitutional isomers from thio-succinimide hydrolysis. EAD fragmentation also provided additional evidence on structural elucidation of disulfide linked products, by preferentially cleaving disulfide bond while leaving peptide backbone intact. The LC-MRM approach with orthogonal fragmentation methods offered complementary information for characterizing AZD8205 biotransformation products in detail. This work, in concert with other studies of ADC biotransformation, creates the necessary scholarship for future implementation of knowledge-based biotransformation pathways predicted by software coupled with LC-MRM analyses to examine a much wider range of potential biotransformation species. This would further uncover structural changes for ADCs in vivo, helping scientists better understand their stability determinants and pharmacokinetics.