Immune checkpoint inhibitors (ICIs), such as anti-PD-1 and anti-PD-L1 Abs, have shown efficacy for the treatment of various cancers. Although research has actively sought to develop new ICIs and immunomodulators, no efficient in vitro assay system is available to evaluate their functional activities. In the present study, we established a two-round MLR with human PBMCs for evaluation of the T cell-activating capacity ofanti-PD-1 and other immunomodulators. We initially performed conventional MLR for this purpose. However, anti-PD-1 blocking Abs could not increase the proliferation of allo-reactive T cells in conventional MLR because PD-L1(+) and PD-L2(+) cells disappeared gradually during MLR. Therefore, we re-applied the same stimulator PBMCs to the allo-stimulated responder cells as a second-round MLR on day 6 when anti-PD-1 or immunomodulators were also added. In this two-round MLR, the proliferation ofallo-reactive T cells was enhanced by anti-PD-1 in a dose-dependent manner or by immunomodulators, such as lenalidomide and galunisertib, a TGF-beta receptor-1 inhibitor. Proliferation was further increased by the combination of immunomodulators with anti-PD-1. Here, we established a modified two-round MLR method with human PBMCs for evaluation of the functional activities ofanti-PD-1 and immunomodulators.