In a clinical diagnostic microbiology laboratory, the current method of identifying bacterial isolates is based mainly on phenotypic characteristics, such as the growth pattern on different media, colony morphology, Gram stain, and various biochemical reactions. These techniques collectively allow high-level accuracy in identifying most bacterial isolates, but they are costly and time-consuming. In our clinical microbiology laboratory, we prospectively assessed the ability of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to identify bacterial strains that were routinely isolated from clinical samples. Bacterial colonies obtained from a total of 468 strains of 92 bacterial species isolated at the Department of Clinical Laboratory at Chiba University were directly placed on target MALDI plates, followed by the addition of CHCA matrix solution. The plates were then subjected to MALDI-TOF MS measurement, and the microorganisms were identified by pattern matching by the libraries in the BioTyper 2.0 software. The identification rates at species and genus levels were 91.7% (429/468) and 97.0% (454/468), respectively. MALDI-TOF MS is a rapid, simple, and high-throughput proteomic technique for the identification of a variety of bacterial species. Since colony to colony differences and the effects of culture duration on the results are minimal, it can be implemented in a conventional laboratory setting. Although for some pathogens, the preanalytic processes should be refined and current database should be improved to obtain more accurate results, the MALDI-TOF MS-based method generally performs as well as the conventional methods and is a promising technology in clinical laboratories.