We evaluated the performance of the Vitek MS for identification of Gram-positive cocci routinely isolated in clinical microbiology laboratories. With a total of 424 well-characterized isolates, the results of the Vitek MS were compared to those of conventional methods and 16S rRNA gene sequencing. The Vitek MS correctly identified 97.9% of the isolates tested to species level. The Vitek MS correctly identified the species of 97.2% of the staphylococci (95.9% of coagulasenegative staphylococci), 97.8% of the streptococci, and 100% of the enterococci. For the identification of Gram-positive cocci isolates, the overall concordance rate between conventional identification and the Vitek MS was 94.5 %. The Vitek MS matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) system can be a reliable and rapid method for the identification of most relevant Gram-positive cocci. In addition, expanding the database of the Vitek MS, especially for coagulase-negative staphylococci, is needed to enhance the performance of the Vitek MS.