Sequencing of anti-vancomycin monoclonal antibody (mAb) Fab region (48,000 Da) was carried out using liquid chromatography-electrospray ionization ion trap mass spectrometry (LC/ESI-MS). Comprehensive strategies were employed to ensure complete sequence coverage. The sequence information was obtained from the spectra of collision-induced dissociation (CID) (MS/MS) of the protonated proteolytic peptides resulting from multiple enzymatic digestions of reduced/S-carboxymethylated (RCM) light chain and Fd fragment. Database searching of the spectra against the published immunoglobulin G (IgG) sequences allowed the identification of all the peptides in constant domains as well as partial sequences in variable domains. The rest of the sequences were deduced by manual interpretation of the peptide tandem mass spectrometry (MS/MS) spectra. The analysis showed that the N-terminus of the heavy chain was modified by the conversion of a glutamine residue to pyroglutamic acid.