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ABSTRACTMalasseziais a genus of lipid-dependent yeasts. It is associated with common skin diseases such as pityriasis versicolor and atopic dermatitis and can cause systemic infections in immunocompromised individuals. Owing to the slow growth and lipid requirements of these fastidious yeasts, convenient and reliable antifungal drug susceptibility testing assays for Malasseziaspp. are not widely available. Therefore, we optimized a broth microdilution assay for the testing of Malasseziathat is based on the CLSI and EUCAST assays for Candidaand other yeasts. The addition of ingredients such as lipids and esculin provided a broth medium formulation that enabled the growth of all Malasseziaspp. and could be read, with the colorimetric indicator resazurin, by visual and fluorescence readings. We tested the susceptibility of 52 strains of 13 Malasseziaspecies to 11 commonly used antifungals. MIC values determined by visual readings were in good agreement with MIC values determined by fluorescence readings. The lowest MICs were found for the azoles itraconazole, posaconazole, and voriconazole, with MIC90values of 0.03 to 1.0 µg/ml, 0.06 to 0.5 µg/ml, and 0.03 to 2.0 µg/ml, respectively. All Malasseziaspp. were resistant to echinocandins and griseofulvin. Some Malasseziaspp. also showed high MIC values for ketoconazole, which is the most widely recommended topical antifungal to treat Malasseziaskin infections. In summary, our assay enables the fast and reliable susceptibility testing of Malasseziaspp. with a large panel of different antifungals.