Differentiation of Methicillin-sensitive Staphylococcus aureus (MSSA), heterogeneous Vancomycin-Intermediate S. aureus (hVISA) and Vancomycin-Intermediate S. aureus (VISA) is important for proper therapy and timely intervention of outbreaks. In this study, a total of 90 S. aureus isolates were enrolled, including 21 MSSA, 25 VSSA, 29 hVISA and 15 VISA. The protein biomarkers for distinguish among MSSA, hVISA and VISA strains were discovered by label-free DIA quantitative proteomics. In the label-free DIA results, 8786 non-redundant peptides, corresponding to 418 different annotated non-redundant proteins were identified. 5 proteins were identified as potential biomarkers with high sensitivity and specificity for the separate identification of MSSA, hVISA and VISA strains.