OBJECTIVE: We evaluated two SDS-based preparation methods for direct identification of yeasts using MALDI-TOF MS analysis and antifungal susceptibility testing (AFST). METHODS: A total of 149 residual monomicrobial blood culture bottles (BCs) were included. For direct identification via MALDI-TOF MS analysis from positive BCs, two in-house methods with or without a 3-hr incubation step were evaluated. 55 samples were also prepared using a Sepsityper kit. In addition, to investigate the effects of differences in RESULTS: Correct identification rate was 65.8% for the saponin+0.5% SDS method and 70.5% for the 20% SDS after 3-hr incubation, respectively. For the 55 samples evaluated using the Sepsityper kit, both in-house methods showed significantly higher identification rates. For the simulated samples, CONCLUSION: Our newly developed preparation method using 20% SDS demonstrated superior performance to a commercial kit, and can therefore be used for direct identification and AFST.