We studied the usefulness of flow cytometry for detection of vancomycin resistance in Enterococcus faecalis by direct binding of commercially available fluorescent vancomycin to cells obtained from culture. The cells were stained with Vancomycin@FL, sonicated and additionally stained with propidium iodide (PI). Regarding to inductive mechanism of vanA-mediated vancomycin resistance, resistant reference strain was also pre-incubated with vancomycin. PI staining divided cells into two subpopulations. There were significantly lower mean FL1 fluorescence values and mean fluorescence per particle (FL1/FSC) in reference vancomycin-resistant strain than in reference and clinical strains sensitive to this antibiotic. Pre-incubation with vancomycin of vancomycin resistant enterococci strain modified Vancomycin@FL binding, however, cells remained easy to differ. We have demonstrated new, quick and sensitive method for detection of vancomycin resistant strains of E. faecalis. The study proved possibility of detection of vancomycin resistance caused by presence of vanA gene by staining cells with Vancomycin@FL. Flow cytometry approach study of E. faecalis vancomycin resistance by detection of Vancomycin@FL binding to the bacterial cells.