BackgroundRapid and accurate microbial identification and antimicrobial susceptibility testing (AST) are essential for timely use of appropriate antimicrobial agents for bloodstream infection. To shorten the time for isolating colonies from the positive blood culture, various preparation methods for direct identification using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) system were developed. Here, we evaluated the SepsiPrep kit (ASTA Corp.) for direct identification of microorganisms and AST from positive blood cultures using MicroIDSys Elite MALDI‐TOF MS system (ASTA Corp.) and VITEK‐2 system (bioMérieux).MethodsFor direct identification, a total of 124 prospective monomicrobial positive blood culture bottles were included. For direct identification, the pellet was prepared by centrifugation and washing twice. For direct AST, the pellet was suspended in 0.45% saline and adjusted to McFarland 0.5. The results from the direct identification and AST using MicroIDSys Elite and VITEK‐2 system were compared to those from the conventional method performed with pure colony subcultured on agar plate.ResultsCompared to the conventional method using pure colony, correct direct identification rate was 96.5% and 98.5% for 57 gram‐positive isolates and 67 gram‐negative isolates, respectively. For direct AST, among the 55 gram‐positive isolates, the categorical agreement (CA) for staphylococci, streptococci, and enterococci was 96.7%, 98.4%, and 94.1%, respectively. For 66 gram‐negative isolates, the CA for Enterobacterales and non‐fermentative gram‐negative rods was 99.0% and 96.6%, respectively.ConclusionsThe SepsiPrep kit was easy to use combined with MicroIDSys Elite and VITEK‐2 system and also, the correct identification and AST rate were very high.