To evaluate the performance of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in identifying Staphylococcus aureus and in determining its methicillin resistance, we analyzed 76 S. aureus clinical isolates using a linear MALDI-TOF MS. Spectral profile data obtained were compared with the database provided with the instrument, and 74% of the isolates were identified as S. aureus, as confirmed by a nuc-based PCR test. The determination of the methicillin resistance in S. aureus is based on the fact that the spectral profiles of methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) differ greatly from each other. Replicate spectral profiles obtained from each isolate were combined to be a representative spectrum of it, and representative spectral profiles from all the isolates constitute a user's self-established database. All the spectral profiles in the database were classified into two groups based on clustering analysis, and one is for MSSA and another MRSA. There was a little discrepancy between the results from MALDI-TOF MS and from PCR. Seven isolates that are negative for the mecA gene by PCR were identified as MRSA by MALDI-TOF MS. The discrepancy may be partially explained by the heterogeneous nature of methicillin resistance in S. aureus. Our results suggested that comparison of MALDI-TOF MS spectral profiles of microorganism could serve as a simple and rapid method for bacterial identification and antibiotic susceptibility analysis.