We report here that modifications of the preanalytical steps of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identification of yeasts, with regard to the original protocol provided by the manufacturers, appear to be efficient for the reliable routine identification of clinical yeast isolates in medical laboratories. Indeed, when one colony was sampled instead of five and the protein extraction protocol was modified, the performance of MALDI-TOF MS was superior to that of the API ID 32C method (discrepancies were confirmed by using molecular identification), allowing the correct identification of 94% of the 335 clinical isolates prospectively tested. We then demonstrated that the time for which the primary cultures were preincubated on CHROMagar did not impact the identification of yeasts by MALDI-TOF MS, since 95.1 and 96.2% of the 183 clinical yeast isolates prospectively tested were correctly identified after 48 and 72 h of preincubation, respectively