Vancomycin resistance is conferred upon vancomycin-resistant enterococci (VRE) through the replacement of peptidoglycan (PG) stem terminal d-Ala-d-Ala with d-Ala-d-Lac. The d-Ala-d-Lac incorporation can affect both the fitness and virulence of VRE. Here we comprehensively investigate the changes to PG composition in vancomycin-resistant Enterococcus faecalis following the growth in presence of vancomycin using liquid chromatography-mass spectrometry. Using high-resolution mass spectrometry, 104 unique muropeptides fragments were identified and the relative abundance of each fragment was accurately quantified by integrating the ion current of a selected ion using extracted-ion chromatogram. The analysis indicates reduced PG cross-linking, increased carboxypeptidase activities, increased N-deacetylation, and increased O-acetylation in VRE when grown in the presence of vancomycin. We found that O-acetylation preferentially occurred on muropeptides fragments with reduced cross-linking with a pentapeptide stem that terminated in d-Ala-d-Lac. These findings show that O-acetylation preferentially occurred in regions of the cell wall with reduced PG cross-linking on PG units that have stems terminating in d-Ala-d-Lac, serving as markers to prevent both the PG-stem modification by carboxypeptidases and the cell wall degradation by autolysins. Accurate quantitative PG composition analysis provided compositional insights into altered cell wall biosynthesis and modification processes in VRE that contribute to lysozyme resistance and enhanced virulence for VRE grown in the presence of vancomycin.