Introduction: Lymphocytes belong to the leukocyte lineage and include three subpopulations, T and B lymphocytes and NK cells. T and B lymphocytes belong to acquired immunity while NK cells have the characteristics of innate immunity. Morphologically, the cell populations mentioned above do not differ. NK cells do not need matching with MHC molecules for cytolytic activity against extraneous antigen. Leukocyte differentiation antigens determine cell lineage and stage of maturity, and are called CD antigens. NK cells are CD3- and CD16+56. By using the methods of immunofluorescence, CD antigens are marked with anti-CD antibodies to which fluorochromes are bound. Flow cytometry is a diagnostic method that allows immunophenotyping of cells based on cell markers (CD markers). By flow cytometry, with the use of immunophenotyping, it is possible to determine NK cells in peripheral blood, ie the proportion within lymphocytes and the absolute number in blood. The flow cytometry results are most often presented in the form of cytograms. Objective: The purpose of this study was to determine the absolute number of NK cells from a peripheral blood sample using the flow cytometry methods Materials and methods: A sample of one patient admitted to the Department of Medical Laboratory Diagnostics of the Clinical Hospital Center Split (KBC Split) was processed. After the analysis on a hematology counter, there was processing of the sample by staining with fluorescently labeled antibodies to CD45, CD3 and CD16+56, followed by lysis and washing of erythrocytes. The processed sample was analyzed on a FACS Calibur flow cytometer. Results: By the methods of data analysis obtained by flow cytometry from three selected appropriate cytograms, percentage of lymphocytes within leukocytes and the percentage of NK cells within the lymphocyte population were obtained. The absolute number of NK cells was obtained by mathematical calculation using the number of leukocyte measured on the Sysmex hematology analyser XN-1000. Conclusion: Immunophenotyping method should be used to determine NK cells in peripheral blood. The absolute number and the percentage of NK cells determined in the analysed sample were within reference interval.