The thio-succinimide rings resulted from thiol-maleimide reaction connect the antibody backbone and linker-payloads. The thio-succinimide ring has been reported to undergo ringopening hydrolysis. Previously, we confirmed this major biotransformation pathway on both Lc and Hc by using intact mass LC-HRMS. Here, we also monitor the linker-payload hydrolysis through bottom-up LC-MRM approach. Figures show the percentage ratio between nonhydrolyzed and hydrolyzed thio-succinimide for GEC from Lc, SCDK and THT from Hc, respectively. They suggest that over 95% of thio-succinimide was hydrolyzed across all three tryptic products after 72 hr plasma incubation. This finding aligns with our reported observation on the same ADC using intact mass LC-HRMS approach. Both GEC and SCDK tryptic products show 1 hydrolysis product, whereas THT shows 2 hydrolysis products due to two conjugated linker-payload. ) and a 10-ppm window.