Recently, MALDI-TOF MS devices designed for use in clinical laboratories have been commercially introduced in various large centres worldwide. All published studies conclude that MALDI-TOF MS can be implemented easily for routine identification of bacteria and yeasts in a clinical microbiological laboratory. Although all data show that MALDI-TOF MS correctly identifies the great majority of isolates processed routinely, it cannot yet identify every such isolate. Until today, MALDI-TOF MS is inappropriate for the identification of Shigella species, pneumococci and viridans streptococci. Database upgrades and sample enrichment are essential elements to refine the MALDI-TOF MS technique, allowing the method to increase its power. For the identification of a significant proportion of yeasts, an extraction method prior to analysis in the mass spectrometer is mandatory to obtain appropriate spectra. Because of the low marginal costs, and the extreme speed of MALDI-TOF MS, the technique can improve laboratory efficiency when used early in identification protocols. Lengthier, more labour-intensive, and costlier techniques can be reserved for the minority of isolates not identified with high confidence by MALDI-TOF MS. MALDI-TOF MS also has the potential to directly identify pathogens in biological fluids, such as urine samples and blood cultures. For this application however, further well-designed prospective studies are warranted. The potential for identification at the serotype or strain level, and antibiotic resistance profiling within minutes make MALDI-TOF mass spectrometry an ongoing revolution in the clinical microbiology laboratory.