Previously, we reported AZD8205 major biotransformation species such as thio-succinimide linker hydrolysis and minor linker-payload deconjugation followed by thiol group adduct and disulfide bond formation through intact LC-HRMS. These previously reported biotransformation pathways demonstrated the structure-stability determinants of AZD8205 conjugation. Herein we employed bottom-up LC-MS/MS using two different fragmentation approaches to confirm previously observed biotransformation products and reveal further structural details of AZD8205 biotransformation species.