ADC is a fast-growing biotherapeutic that precisely delivers cytotoxic payload to targeted cells. As recently, ADCs have shown great promise in oncology, and there is an increasing interest to expand the development of ADCs in diverse mechanisms of action or other therapeutic areas. ADC is composed of three parts, antibody backbone, linker and payload. The antibody binds to target molecules and directs the delivery of the payload. The payload works against one of the two cellular functions, microtubule-dependent mitosis or DNA replication, to induce cell apoptosis. Due to the acute toxicity of the payload, ADCs are designed to remain intact during circulation, to avoid off-target toxicity. However, in vitro or in vivo biotransformation of ADCs may occur possibly because of the presence of enzymes or variations in redox potentials. Most importantly, each individual ADC may undergo distinctive biotransformation pathways, largely defined by linker design, conjugation sites and methods, and payload properties. Thus, it is essential to characterize the biotransformation products of ADCs in detail to understand their stability and pharmacokinetic properties. In the past few years, researchers investigated ADC biotransformation from various perspectives. The biotransformation of ADCs could stem from linker hydrolysis, antibody modification, linker-payload deconjugation and payload metabolism. Previously, we reported an unexpected deamidation reaction occurring in the complementarity-determining regions (CDRs) of the antibody backbone of an ADC, MEDI7247. The change from asparagine to aspartic acid leads to decreased binding affinity between the ADC and its target protein, thus compromising its overall efficacy. Su et al. reported the linker-payload deconjugation from an anti-HER2 THIOMAB ADC. The premature release of payload could result in decreased efficacy and undesired cytotoxicity. They also reported the tubulysin payload from a THIOMAB ADC went through a deacetylation reaction, leading to inactive payload. Such a biotransformation could compromise its efficacy.