eWe developed an in-house assay for the direct identification, by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, of yeasts in blood culture. Sixty-one representative strains from 12 species were analyzed in spiked blood cultures. Our assay accurately identified 95 of 107 (88.8%) positive blood cultures and outperformed the commercial Sepsityper kit (81.7% identification). P rompt, appropriate, first-line antifungal treatment has been shown to improve outcomes in patients with fungemia, but identification is often delayed by the need for subculture from positive blood cultures (1, 2). Mass spectrometry (MS) techniques based on matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) technology have made it possible to identify species directly from positive blood cultures (3). The sample contains human cells, and identification protocols must lyse these cells efficiently (without disrupting the microorganism) and eliminate the unwanted human proteins. In-house protocols based on the lytic agent saponin have been described for bacterial identification (4, 5), but only a few assays have been developed for yeastpositive blood cultures (6-9). Bruker produces an easy-to-use kit, Sepsityper, which has yielded good results for the characterization of both bacteria and yeasts (10, 11). However, this commercial test has never been compared, for yeast identification, with any inhouse protocol. We developed an in-house protocol for the direct identification, by MALDI-TOF MS, of the yeast species commonly isolated from blood cultures. We compared the results obtained by our assay to those obtained by Sepsityper.To facilitate the testing of large numbers of strains and species (12), we used blood collected from healthy volunteers and spiked the blood culture flasks with cultured yeasts. We used 61 strains formerly isolated in the context of invasive fungal infection (stored at -80°C) and previously identified by MALDI-TOF MS.