Low or absent natural killer (NK) cell activity is included as one of the HLH-2004 diagnostic criteria. To improve the diagnosis of HLH, we aimed to establish a rapid and reliable NK cell activity assay that avoids the use of radioactivity. The K562 cell line, as standard NK target cells, was engineered to stably express enhanced green fluorescent protein (EGFP), which can be quantified by flow cytometry. The EGFP-flow cytometry method for measuring NK cell activity was improved by double staining of early and late apoptotic target cells. Whole-blood samples from healthy volunteers were assessed with this method, which demonstrated that optimal conditions were effector-target ratio of 10:1 and incubation time of 4 h. This method was further evaluated for samples from 113 HLH patients and 64 healthy volunteers. Mean NK cell activity in either primary or secondary HLH patients was significantly lower (P < 0.001) than in healthy individuals (20.23 ± 4.12%). Furthermore, primary HLH patients (10.76 ± 2.54%) exhibited even lower (P < 0.001) NK cell activity compared with secondary HLH patients (15.01 ± 3.62%). We have optimized and implemented this method in clinically relevant samples.