Abstract: Among different mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) has become popular in infectious disease management, infection control and antimicrobial stewardship especially in the field of bacterial and fungal diseases, due to its simple sample pre-treatment, rapid identification of microorganism, and future scope of detecting biomarkers in infectious diseases, antimicrobial susceptibility testing, epidemiological typing, microbial virulence factors and its use in the field of virology and parasitology as well. The technique has the potentiality for point-of-care diagnosis and screening of large number of samples simultaneously. Many laboratories across globe use this technique in routine microbiology laboratory due to its efficiency and cost-per-analysis point of view. It outcompetes the classical microbial identification methods. FDA approved both VITEK MS system (BioMérieux) and MALDI_TOF MS Biotyper (Bruker Daltonics) for microbial identification. The identification system requires pure culture (∼108microbial cells) of a single species bacteria, mycobacteria or fungi from liquid or solid media. The mixed microbial species are hard to identify unless the ratio between the species is not lower than 1:5. For Mycobacteria, Nocardia, and fungi a cell destruction or extraction step is required. The majority studies reported correct identification >95% isolates. However, for better identification, periodic update of database is essential. Optimization of the distinction of few species still remains problematic like E. coli and Shigella species. The other applications of MALDI that may have impact on management of infectious diseases include antimicrobial susceptibility testing and epidemiological typing. Antimicrobial susceptibility testing targets a) modification and degradation product of antimicrobial after exposure to microbe, b) proteomic changes in resistant versus susceptible microbial species, c) shift of protein peak after growth of microbe in presence of antimicrobials, d) semi-quantitative estimation of intracellular/extracellular antimicrobials. The techniques require further standardization before getting regulatory approval. In case of microbial typing, the technique compares unique isolate peaks, whereas the identification targets universally conserved peaks. Modification of MALDI-TOF like SELDI-TOF (Surface-enhanced laser desorption) or ESI-MS (Electron spray ionization) are currently explored for their clinical potential.