Thiol-maleimide conjugation is a highly popular reaction due to its specificity, selectivity and fast kinetics. In recent decades, this reaction has been used in various fields, including biomolecule labelling and synthesis of novel polymeric materials. One of the most important applications of the thiol-maleimide reaction is the bioconjugation between linker-payload and antibody backbone, to generate antibody-drug conjugates (ADCs). Comparing to welldeveloped small molecule drugs, ADC shows promise as a new class of therapeutics, with the first ADC approved in 2001. As of October 2023, there are 15 different ADCs approved, and 10 of them utilize thiol-maleimide reaction during their syntheses. There are mainly two competitive biotransformation processes occurring at the thio-succinimide site from linker. The reversible Retro-Michael reaction of thio-succinimide leads to the premature linker-payload deconjugation. Different approaches have been tested to avoid such problem. On the other hand, the thio-succinimide is also subject to ring opening hydrolysis, and its product prevents the deconjugation, increasing the stability of ADCs. Notably, the hydrolysis of thio-succinimide results in a pair of isomeric products, thio-aspartyl (thio-Asp) and thio-isoaspartyl (thio-isoAsp). This finding was confirmed by observations from several analytical techniques, including FTIR and NMR. Nevertheless, to our knowledge, the differentiation of these isomers using LC-MS has never been reported. Biotransformation of ADCs is predominantly characterized by intact protein mass analysis through liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS). Previously, we reported a workflow that uses hybrid ligand-binding assay (LBA) coupled with intact protein mass LC-HRMS to study ADC drug-antibody ratio (DAR) changes resulted from biotransformation. However, the isomeric structures cannot be distinguished through the intact protein mass analysis due to their exact same mass. Furthermore, the reverse phase LC (RPLC) may not be sufficient to separate large molecules with such subtle structural difference. Other than intact mass analysis, researchers also synthesize reference materials and compare their retention time (RT) to identify isomers. The complicated synthesis and purification processes compromise the efficiency of this analytical approach. Here, we report a bottom-up LC-MS/MS approach with the help from newly developed electron-activated dissociation (EAD) to distinguish thio-succinimide hydrolysis isomeric products.