Here, we report a digestion-free middle-down mass spectrometry (DF-MDMS) method for absolute quantification of several ADCs on a ZenoTOF 7600 mass spectrometer (SCIEX). Collision-Induced Dissociation (CID) was used for absolute quantification of ADCconjugated payload. We applied immunocapture to extract ADCs from biological matrices, followed by acid elution from beads and optional reduction and deglycosylation. The results demonstrate that both cleavable and non-cleavable linker ADCs can be quantified using this DF-MDMS method with sufficient sensitivity. This method can facilitate ADC quantification, especially high-throughput analysis of non-cleavable linker ADCs.