In conclusion, we successfully established a novel digestion-free analytical method for absolute quantification of conjugated payload from ADCs using CID-based mass spectrometry. Compared to other LCMS-based methods, this approach is cost and timeeffective, which significantly reduces the workload on enzymatic digestion and downstream data processing. It has been applied to multiple ADCs, including the noncleavable linker ADC trastuzumab emtansine. The results demonstrated that various types of ADCs can be quantified with reasonable sensitivity and straightforward data processing. We believe that this method can improve the throughput of bioanalysis, especially for non-cleavable linker ADCs. It can also simplify analytical chemistry workflows for ADCs in samples with pure buffer where no immunocapture and direct MS injection may be applied.