With this method, a single injection was sufficient to characterize the amino acid sequence with complete sequence coverage. In addition, glycosylation and drug-loaded peptides could be identified from MS/MS spectra. A drug-loaded peptide fragmentation mass spectra study yielded drug-specific fragments, which reinforced the confidence about the identifications. The results reveal the ability of the sheathless CZE–MS/MS method to characterize an ADC’s primary structure in a single experiment.