An interlaboratory study using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to determine the identification of clinically important yeasts (n ‫؍‬ 35) was performed at 11 clinical centers, one company, and one reference center using the Bruker Daltonics MALDI Biotyper system. The optimal cutoff for the MALDI-TOF MS score was investigated using receiver operating characteristic (ROC) curve analyses. The percentages of correct identifications were compared for different sample preparation methods and different databases.