<title>Abstract</title> Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) is now used as a routine technique for the fast and reliable identification of fungi at the species level and, currently, it represents an important phenotypic methodology based on proteomic profiles. The main limitations to MALDI-TOF MS for fungal identification are related to sample quality (e.g. quality of biological material such as rigidity or pigmentation of cell walls), sample preparation (e.g. the myriad of sample preparation methodologies that deliver different data sets to different MALDI-TOF MS databases) and the databases themselves (e.g. the 'black-box' commercial databases). This chapter presents an overview and discussion of the use of MALDI-TOF MS for fungal identification. The major known limitations of the technique for fungal taxonomy, and how to overcome these, are also discussed.