Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) represents one of the most accurate, reliable, and fast methods for the identification of bacterial strains from positive cultures, and therefore it has largely replaced all other previously used approaches for microbial identification. The main application of MALDI-TOF MS in clinical microbiology laboratories is the identification of bacteria from colonies recovered from solid culture media. This chapter discusses specific identification procedures that are needed for some bacteria, such as Actinomycetes and Mycobacteria. The performance of MALDI-TOF MS identification relies on the number of mass spectra that reach the quality allowing identification and the number of correct identifications. MALDI-TOF MS has also been proposed for Staphylococcus aureus strain typing or for the detection of biomarkers of the most virulent toxigenic isolates. MALDI-TOF MS could also be used for Mycobacterium.