Advances in mass spectrometry have enabled the investigation of various biological systems by directly analyzing diverse sets of biomolecules (i.e., proteins, lipids, and carbohydrates), thus making a significant impact on the life sciences field. Over the past decade, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been widely utilized as a rapid and reliable method for the identification of microorganisms. MALDI-TOF MS has come into widespread use despite its relatively low resolving power (full width at half maximum, FWHM: < 5,000) and its incompatibility with tandem MS analysis, features with which other high-resolution mass spectrometers are equipped. Microbial identification is achieved by searching databases containing mass spectra of peptides and proteins extracted from microorganisms of interest, using scoring algorithms to match analyzed spectra with reference spectra. In this paper, we give a brief overview of the diverse applications of rapid and robust MALDI-TOF MS-based techniques for microbial identification in a variety of fields, such as clinical diagnosis and environmental and food monitoring. We also describe the fundamental principles of MALDI-TOF MS. The general specifications of the two major MS-based microbial identification systems available in the global market (BioTyper® and VITEK® MS Plus) and the distribution of these instruments in Republic of Korea are also discussed. The current review provides an understanding of this emerging microbial identification and classification technology and will help bacteriologists and cell biologists take advantage of this powerful technique.