Matrix-Assisted Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) has revolutionized the workflow in clinical microbiology laboratories by providing rapid and accurate identification of bacteria and fungi, reducing the time to obtain microbiological diagnoses by 24 hours compared to conventional methods. MALDI-TOF MS is based on the proteomic analysis of microbial protein profiles, offering high sensitivity, throughput, and simplicity. It has been widely applied to positive blood cultures for microorganism identification and antimicrobial resistance detection, as well as to other areas such as antibiotic susceptibility testing, aminoacidic sequence identification, and microbial typing. Despite its advantages, MALDI-TOF MS has limitations, particularly in distinguishing closely related species and identifying certain types of bacteria and fungi. The implementation of a comprehensive database with reference spectra is crucial for improving the reliability and accuracy of MALDI-TOF MS. Recent advancements in automation have further streamlined the sample preparation process, reducing human errors and enhancing the reproducibility of results. MALDI-TOF MS has also shown promise in identifying pathogens from clinical samples such as urine and cerebrospinal fluid, although further studies are needed to validate these applications. Overall, MALDI-TOF MS is a valuable tool in clinical microbiology, offering rapid and reliable identification of microorganisms and antimicrobial resistance markers.Matrix-Assisted Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) has revolutionized the workflow in clinical microbiology laboratories by providing rapid and accurate identification of bacteria and fungi, reducing the time to obtain microbiological diagnoses by 24 hours compared to conventional methods. MALDI-TOF MS is based on the proteomic analysis of microbial protein profiles, offering high sensitivity, throughput, and simplicity. It has been widely applied to positive blood cultures for microorganism identification and antimicrobial resistance detection, as well as to other areas such as antibiotic susceptibility testing, aminoacidic sequence identification, and microbial typing. Despite its advantages, MALDI-TOF MS has limitations, particularly in distinguishing closely related species and identifying certain types of bacteria and fungi. The implementation of a comprehensive database with reference spectra is crucial for improving the reliability and accuracy of MALDI-TOF MS. Recent advancements in automation have further streamlined the sample preparation process, reducing human errors and enhancing the reproducibility of results. MALDI-TOF MS has also shown promise in identifying pathogens from clinical samples such as urine and cerebrospinal fluid, although further studies are needed to validate these applications. Overall, MALDI-TOF MS is a valuable tool in clinical microbiology, offering rapid and reliable identification of microorganisms and antimicrobial resistance markers.