Methicillin-resistant Staphylococcus aureus (MRSA) is the leading cause of fatal bacterial infections in hospitals and has become a global health threat. Although the resistance mechanisms of beta-lactam antibiotics have been studied for decades, there are few attempts at systems-wide investigations into how the bacteria respond to antibiotic stress. Spectral counting-based label-free quantitative proteomics has been applied to study global responses in MASA and methicillin-susceptible S. aureus (MSSA) treated with sub-inhibitory doses of oxacillin, a model beta-lactam antibiotic. We developed a simple and easily repeated sample preparation procedure that is effective for extracting surface-associated proteins. On average, 1025 and 1013 proteins were identified at a false discovery rate threshold of 0.01, for the untreated group of MRSA and MSSA. Upon treatment with oxacillin, 81 proteins (65 up-regulated, 16 down-regulated) were shown differentially expressed in MRSA (p