In contrast to the conventional LC-MS-based bioanalysis method to measure ADC with cleavable linker that requires enzymatic digestion to release the payload, DF-MDMS quantification uses CID to quantify the payload from ADC without enzyme digestion. The overall workflow is shown in Figure . ADCs were immunocaptured by magnetic beads conjugated with capture antibody and eluted with acid, followed by optional reduction if inter-chain disulfide bonds exist and/or deglycosylation when needed, especially for heterogenous ADCs whose spectra can be complicated further by their glycosylation profiles. This further breaks down the intact ADC into single chains, which could be separated chromatographically, reducing complexity and enabling higher resolution for quantification. The extracts were injected into LC coupled with SCIEX ZenoTOF 7600 system. Optimized collision energy was applied to dissociate the payload, which permits ADC quantification using signature payload MS 2 product ions. To demonstrate the application of the DF-MDMS quantification method, four different ADCs in biological matrices were examined: three cleavable linker ADCs with TOP1i payload, and one non-cleavable linker ADC trastuzumab emtansine. The parameters and assay results of each ADC are summarized in Table , including molecule and sample preparation details, data acquisition parameters, assay dynamic range, etc. The overall results show that assays successfully met accuracy acceptance criteria at ±20 % / 25 % for LLOQ; ≥75 % of standards and ≥66.7 % of QCs meet acceptance criteria. Correlation coefficient (r) for standard curves was ≥0.985. The assay setup and parameter optimization are discussed in the following sections.