Thio-succinimide hydrolysis is analogous to protein deamidation. The asparagine deamidation generates a succinimide intermediate before the addition of water. The resulting aspartic acid results in ~ 1 Da molecular weight increase compared to its original mass. Similar to thiosuccinimide hydrolysis, the asparagine deamidation also generates a pair of isomeric products, aspartic acid, and iso-aspartic acid. We previously reported the identification of asparagine deamidation at the complementarity-determining regions of another ADC, MEDI7247, employing LC-MS/MS. The diagnostic ions were c+57 and z-57 coming from the iso-aspartic acid after dissociation between α-and β-carbons through EAD. Here, we investigated the feasibility of employing similar strategy (+57 u or -57 u related signature ions) to differentiate thio-Asp and thio-isoAsp, despite the slight structure difference compared to aspartic acid and iso-aspartic acid. Moreover, we further explored if EAD could generate more unique diagnostic ions only for thio-succinimide hydrolysis products. To our knowledge, we are the first to report an in-depth characterization of thio-succinimide hydrolysis isomerization using a bottom-up LC-MS/MS approach without requiring reference materials.