Oritavancin is a lipoglycopeptide antibiotic that exhibits potent activities against vancomycin-resistant Gram-positive pathogens. Oritavancin differs from vancomycin by a hydrophobic side chain attached to the drug disaccharide, which forms a secondary binding site to enable oritavancin binding to the cross-linked peptidoglycan in the cell wall. The mode of action of secondary binding site was investigated by measuring the changes in the peptidoglycan composition of Staphylococcus aureus grown in the presence of desleucyl-oritavancin at subinhibitory concentration using liquid chromatography-mass spectrometry (LC-MS). Desleucyl-oritavancin is an Edman degradation product of oritavancin that exhibits potent antibacterial activities despite the damaged D-Ala-D-Ala binding site due to its functional secondary binding site. Accurate quantitative peptidoglycan composition analysis based on 83 muropeptide ions determined that cell walls of S. aureus grown in the presence of desleucyl-oritavancin showed a reduction of peptidoglycan cross-linking, increased muropeptides with a tetrapeptide-stem structure, decreased O-acetylation of MurNAc, and increased N-deacetylation of GlcNAc. The changes in peptidoglycan composition suggest that desleucyl-oritavancin targets the peptidoglycan template to induce cell wall disorder and interferes with cell wall maturation.IMPORTANCE Oritavancin is a lipoglycopeptide antibiotic with a secondary binding site that targets the cross-linked peptidoglycan bridge structure in the cell wall. Even after the loss of its primary D-Ala-D-Ala binding site through Edman degradation, desleucyl-oritavancin exhibits potent antimicrobial activities through its still-functioning secondary binding site. In this study, we characterized the mode of action for desleucyl-oritavancin's secondary binding site using LC-MS. Peptidoglycan composition analysis of desleucyl-oritavancin-treated S. aureus was performed by determining the relative abundances of 83 muropeptide ions matched from a precalculated library through integrating extracted ion chromatograms. Our work highlights the use of quantitative peptidoglycan composition analysis by LC-MS to provide insights into the mode of action of glycopeptide antibiotics.