Enhanced green fluorescent protein (EGFP) was stably expressed in human erythroleukaemia K562 cells (EGFP-K562) and used as target cells for measurement of natural killer (NK) cell cytotoxicity by flow cytometry. The compromised EGFP-K562 target cells were stained with propidium iodide (PI) and showed dual (green-red) fluorescent. Although the kinetic study demonstrated that the optimal incubation time for the assay was 4 h, a 2-h incubation period also gave comparable results. This new technique correlated strongly with the standard chromium (51Cr) release assay at the correlation coefficients of 0.87 and 0.89 at p-value <0.001 for 2- and 4-h incubation times, respectively. The EGFP-K562 stable cell line provides a novel method to measure NK cytotoxicity by flow cytometry without pre-staining or pre-labeling target cells.