Events including antibody-antigen affinity,internalization,trafficking and lysosomal prote-olysis combinatorially determine the efficiency of antibody-drug conjugate(ADC)catabolism and hence the toxicity.Nevertheless,an approach that conveniently identifies proteins requisite for payload release and the ensuing toxicity for mechanistic studies and quality assessment is lacking.Considering the plethora of ADC candidates under development,we developed a target-responsive subcellular catabolism(TARSC)approach that examines ADC catabolism and probes changes in response to targeted interfer-ences of proteins of interest.We firstly applied TARSC to study the commercial T-DM1 and the bio-similar.We recorded unequivocal catabolic behaviors regardless of the absence and presence of the targeted interferences.Their negligible differences in TARSC profiles agreed with their undifferentiated anti-tumoral efficacy according to further in vitro viability and in vivo tumor growth assays,highlighting TARSC analysis as a useful tool for biosimilarity assessment and functional dissection of proteins requi-site for ADC catabolism.Additionally,we employed TARSC to investigate the catabolic behavior of a new trastuzumab-toxin conjugate.Collectively,TARSC can not only characterize ADC catabolism at(sub)cellular level but also comprehensively determine which protein targets affect payload release and therapeutic outcomes.Future use of TARSC is thus anticipated in early-stage screening,quality assessment and mechanistic investigations of ADCs.