This DF-MDMS quantification is a valuable tool for accurate DAR measurement of ADCs with non-cleavable linker, such as trastuzumab emtansine. Figure shows the payload specific MS2 XIC of various DAR species for light and heavy chain for trastuzumab emtansine. Compared to ADC1 MS2 XIC (Figure ), it shows the existence of multiple species (Figure ) with low intensity due to limited payload dissociation efficiency. Even with the above-mentioned challenges, the trastuzumab emtansine ADC concentration can be quantified using DF-MDMS CID method with a linear dynamic range of 3000-90000 ng/mL for LC1 and 9000-90000 ng/mL for LC2 (light chain-2 payloads). We were not able to establish a calibration curve for heavy chain species as the high heterogeneity of the conjugation site on the heavy chain in trastuzumab emtansine might play a role in the reduced ionization efficiency and consistency of heavy chain species. Optimization of liquid chromatography strategy and alternative mass spectrometry methods should be considered for enabling absolute quantification of heterogeneous ADCs with different structures of payloads using DF-MDMS.