Antibody-drug conjugates (ADCs) require multiple assays to characterize their PK. These assays can separately evaluate the ADC by quantifying the antibody or the conjugated drug and may give different answers due to assay measurement differences, heterogeneous nature of ADCs and potential biotransformations that occur in vivo.We present a new version of the antibody-conjugated drug assay for valine-citrulline-linked monomethylauristatin E (vcMMAE) ADCs. A stable isotope-labeled internal standard, protein A affinity capture and solid-phase cleavage of MMAE using papain was used prior to LC-MS/MS analysis.The assay was used to assess the difference in ex vivo drug-linker stability of native-cysteine versus engineered cysteine ADCs and to determine the number of drugs per antibody of a native-cysteine ADC in vivo.