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Unlike Enterococcus faecium strains, some Enterococcus lactis strains are considered potential probiotic strains as they lack particular virulence and antibiotic resistance genes. However, these closely related species are difficult to distinguish via conventional taxonomic methods. Here, for the first time, we used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with BioTyper and in-house databases to distinguish between E. faecium and E. lactis. A total of 58 reference and isolated strains (89.2%) were correctly identified at the species level using MALDI-TOF MS with in-house databases. However, seven strains (10.8%) were not accurately differentiated as a single colony was identified as a different species with a similar score value. Specific mass peaks were identified by analyzing reference strains, and mass peaks at 10,122 ± 2 m/z, 3650 ± 1 m/z, and 7306 ± 1 m/z were unique to E. faecium and E. lactis reference strains, respectively. Mass peaks verified reproducibility in 60 isolates and showed 100% specificity, whereas 16S rRNA sequencing identified two different candidates for some isolates (E. faecium and E. lactis). Our specific mass peak method helped to differentiate two species, with high accuracy and high throughput, and provided a viable alternative to 16S rRNA sequencing. [ABSTRACT FROM AUTHOR]