Several flow cytometric methods for measuring natural killer cell activity have been developed. Commonly used protocols involve the staining of target cells with various fluorescent dyes. However, these protocols are not applicable to certain experimental settings. Therefore, we used Calcein AM (CAM), which has been reported to be the most suitable dye for use in target cell staining protocols, as a means of developing an improved flow cytometry-based NK cytotoxicity assay involving effector cell staining. Peripheral blood mononuclear cells (PBMCs) isolated by gradient density centrifugation and expanded NK cells were used as effector cells. Cytotoxicity against K562 cells and several hematologic cancer cell lines was measured by a flow cytometry-based method using CAM and propidium iodide. The new assay was compared with a standard 51Cr release assay (CRA) in terms of its ability to measure the cytotoxicity of NK cells in PBMCs and expanded NK cells against K562 cells. The optimal concentration of CAM for staining effector cells was 0.05 μM, and CAM fluorescence intensity in effector cells was maintained for 4 hours. CAM staining had no significant effect on NK cell activity in human PBMCs or expanded NK cells. Comparison of the CRA and this new assay using K562 cells revealed a good correlation (PBMCs, r = 0.894; expanded NK cells, r = 0.887). Distinct separation between target tumor cells (Daudi, Raji, RPMI8226, U266, U937, and K562 cells) and CAM-stained PBMCs (E:T ratio, 12.5:1 to 50:1) or expanded NK cells (E:T ratio, 0.5 to 4:1) was observed after incubation for 1 or 4 hours. In summary, we successfully developed an effective flow cytometry-based assay for assessing the activity of NK cells in PBMCs and expanded NK cells against K562 cells and various types of hematologic cancer cells.