Thiol-maleimide reaction is widely used for bioconjugation purposes. The resulted thiosuccinimide structure is vulnerable to ring-opening hydrolysis. ADC1 is a thio-succinimidelinked ADC, produced from reducing interchain disulfide bonds, followed by covalently conjugating linker-payload to the thiol groups from cysteine side chains using maleimide chemistry. To identify and characterize its hydrolysis isomers, we used a signature ADC1 tryptic digested product, which is composed of a tripeptide GEC, succinimide (with or without water adduct) and R2 to demonstrate the process. Figure shows the hydrolysis reaction, and potential diagnostic ions for differentiating hydrolysis isomers, using GEC+succinimide+R2 as an example.