Fcγ receptors (FcγRs), which bind to the Fc regions of antibodies, play an important role in antibody effector functions. In humans, there are four types of activating FcγRs: FcγRI, FcγRIIa, FcγRIIIa, and FcγRIIIb. These are expressed on various effector cells such as natural killer (NK) cells, neutrophils and macrophages. FcγRIIIa expressed on NK cells is known to play a pivotal role in antibody-dependent cellular cytotoxicity (ADCC) by therapeutic monoclonal antibodies (mAbs). To assess the ADCC activity of mAbs, the killing of target cells is often measured using human peripheral mononuclear blood cells (hPBMCs) or isolated primary NK cells as effector cells. These assays can directly assess the cytotoxicity induced by mAbs, but require fresh blood from donors, and are insufficiently reproducible due to differences in effector cell activity among donors. We developed a cell-based assay using reporter cell lines expressing human FcγR and a nuclear factor of activated T cells (NFAT)-driven luciferase reporter gene (Jurkat/FcγR/NFAT-Luc), which can estimate the activation of various FcγRs by antigen-bound mAbs in vitro, with high reproducibility. The usefulness of this assay was confirmed by comparing mAbs activity with different abilities to activate FcγRs, including Fc-engineered anti-CD20 mAbs and anti-EGFR mAbs with different IgG subclasses. We also confirmed the application of this assay for the characterization of mAbs product-related substances. Our FcγR reporter assay is a promising new tool for the characterization of therapeutic mAbs in various stages of mAbs development.