We have presented a comprehensive profiling approach focused on the in vivo biotransformation pathway for a series cysteine-conjugated of ADCs with differing linkers. We employed immuno-affinity capture enrichment, coupled with LC-HRMS and complemented with LC-MRM confirmation, to obtain characterization data at protein subunit level. The HRMS data were interpreted with an unbiased deconvolution method. Key biotransformation species were identified with intact mass and fragmentation approaches, and relative quantification was performed based on the peak area. The elimination of the parent molecule and generation of the biotransformed species as function of time post-dose was used to map the biotransformation reaction pathway of the ADC molecules. When applying this methodology in concert, to a group of ADCs, the structure-stability relationship was established substantiating the importance of the linker structure for this ADC conjugation approach. These data, along with additional information, reported elsewhere 21 led to the selection AZ14170133 as the optimal linker-payload resulting in AZD8205 ADC. To expand our future understanding of bioconjugate and catabolites interactions with endogenous molecules, we will need to apply this methodology, across bioconjugates with varying conjugation approaches, linkers and payloads. Notably, it will also be important to evaluate biotransformation pathways in various microenvironments either in specific organs/tissues or tumors to fully understand the determinants for their efficacy/safety profile. Eventually, the biotransformation information obtained in animal models/in vitro experiments may be translated to potential patient populations as part of clinical studies.