The widespread outbreak of Methicillin-Resistant Staphylococcus Aureus (MRSA) has caused clinical and epidemiological concern in hospital environment. The emergence of Vancomycin-Intermediate S. Aureus (VISA) and, more recently, Vancomycin-Resistant S. Aureus (VRSA) has further alarmed clinician and scientist worlwide. The objective of this study is to determine the optimum concentration of sample protein from MRSA after treatment with acetone extract from Quercus infectoria gall. Comparison of the Protein Expression Profile (PEP) between the treated MRSA and untreated strain as control was obtained using 2-dimensional gel electrophoresis. The Minimum Inhibitory Concentration (MIC) value of acetone extract of galls from Q. infectoria against two strains of MRSA; ATCC 33591 and PPUKM clinical isolate was determined by broth microdilution method. The MIC value of acetone extracts against both strains of MRSA was 0.3125 mg mL −1 compared to vancomycin (0.00195 mg mL −1 ). The optimum concentration of MRSA protein that produced the best resolution was 100 µg. Manifold technique was observed to produce a gel with better resolution and greater number of spot compared with the strip holder technique. This study showed that there were 7 protein spots that represented the increased in the protein expression of more than 2-fold in the MRSA treated with acetone extract of galls Q. infectoria compared to the untreated group. This preliminary study on the PEP of Q. infectoria galls extract-treated MRSA may provide an insight of its antimicrobial mechanism which could lead to the identification of target protein in the future development of a new effective regimen for the treatment of MRSA infections.