Very recently a novel method for differentiation of bacteria and fungi has been developed, that is, identification by means of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). This differentiation relies on the exact measurement of species-specific protein spectra of ribosomal proteins. It is at least as accurate as conventional biochemical differentiation methods, but provides results within minutes. In addition to differentiation of bacteria and yeasts grown on agar plates, direct identification is feasible from positive blood cultures as well as from urine samples of patients suffering from urinary tract infections. Future developments of MALDI-TOF MS for clinical microbiological purposes include the detection of beta-lactamase and carbapenemase activity as well as genotyping of bacteria below the species level.