Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a rapid, sensitive, and cost-effective tool for microbial identification and diagnosis. This technology uses intact cells or cell extracts to generate peptide mass fingerprints (PMFs), which are compared against a reference database to identify microorganisms. MALDI-TOF MS has been widely adopted by microbiologists for various applications, including microbial identification, strain typing, epidemiological studies, detection of biological warfare agents, and identification of water- and food-borne pathogens. The technology's limitations include the need for a comprehensive spectral database and the inability to identify new isolates if their type strains are not already in the database. Despite these constraints, MALDI-TOF MS has shown high accuracy and reliability in clinical settings, particularly in the diagnosis of bacterial infections, food and water-borne diseases, and environmental samples. It has also been applied in virology and mycology for virus identification, genotyping, and strain typing. The future of MALDI-TOF MS lies in improving database coverage, enhancing reproducibility, and expanding its applications to more complex microbial systems.Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a rapid, sensitive, and cost-effective tool for microbial identification and diagnosis. This technology uses intact cells or cell extracts to generate peptide mass fingerprints (PMFs), which are compared against a reference database to identify microorganisms. MALDI-TOF MS has been widely adopted by microbiologists for various applications, including microbial identification, strain typing, epidemiological studies, detection of biological warfare agents, and identification of water- and food-borne pathogens. The technology's limitations include the need for a comprehensive spectral database and the inability to identify new isolates if their type strains are not already in the database. Despite these constraints, MALDI-TOF MS has shown high accuracy and reliability in clinical settings, particularly in the diagnosis of bacterial infections, food and water-borne diseases, and environmental samples. It has also been applied in virology and mycology for virus identification, genotyping, and strain typing. The future of MALDI-TOF MS lies in improving database coverage, enhancing reproducibility, and expanding its applications to more complex microbial systems.