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ABSTRACT Background Therapeutic antibodies targeting the PD-1/PD-L1 immune checkpoint are widely used in cancer therapy and are under active further development. Historically, the antitumor activity of PD-1/PD-L1 immune checkpoint inhibitors has been evaluated using in vivo and ex vivo test methods; however, a simple in vitro assay method to evaluate antitumor activity accurately is needed for the efficient development of new therapeutic agents. In the present study, we attempted to establish a simple cell-based assay system to evaluate the modulating effect of PD-1/PD-L1 immune checkpoint inhibitors on cytotoxic activity. Methods We established a new natural killer (NK) cell line stably transfected with the PD-1 and IL-2 genes and a new NK-sensitive target cell line stably transfected with the PD-L1 gene. Then, the assay system was established by co-cultivation of the established cell lines and measurement of the cytotoxic activities using the europium release assay. To confirm the performance of the established assay system, model therapeutic antibodies to block the PD-1/PD-L1 signal, nivolumab and atezolizumab were added to the co-culture system and the modulating effect on the cytotoxic activities were evaluated. Results Nivolumab and atezolizumab clearly showed a modulating effect on cytotoxic activity in a dose-dependent manner in our assay system, whereas a human IgG isotype control antibody did not show any modulating effect on the assay system. Conclusion The newly established cell-based assay system can quantitatively evaluate the modulating effect of PD-1/PD-L1 immune checkpoint inhibitors by measuring cytotoxic activity, playing an important role in antitumor effects as innate immunity.