Reduction and deglycosylation are optional procedures in the DF-MDMS quantification workflow. Among four ADCs, ADC1 and ADC2 DAR8 are stochastic cysteine-conjugated ADCs, which means they do not have inter-chain disulfide bonds. Therefore, the acidic condition (during elution and liquid chromatography separation) would disrupt the noncovalent bond connecting the light and heavy chain of the ADC. For trastuzumab emtansine and ADC2 DAR4, the reduction step is necessary to reduce the interchain disulfide bond, generating single antibody chain to improve chromatographic resolution. Optionally, deglycosylation reduces the number of species, collapsing the different glycosylated forms together. Deglycosylation is generally suggested for more heterogeneous ADCs (i.e., ADC2 DAR4 and trastuzumab emtansine), where more varied DAR species contribute to the complexity of spectra. Because of the conjugation approach that employed all interchain disulfides, ADC2 DAR8 and ADC1 are more homogeneous and glycosylation peaks co-elute. Therefore, deglycosylation of these ADCs was not necessary for quantification. Reduction and deglycosylation simplify the mass profile of the heterogeneous ADC and thus increase assay sensitivity. The processed ADC was then analyzed with LC-MS/MS, where payload-specific product ions dissociate from light and heavy chains. The integrated peak area of signature payload product ions was then used for absolute quantification.