More than 100 monoclonal antibodies (mAbs) have been approved by FDA. The mechanism of action (MoA) involves in neutralization of a specific target via the Fab region and Fc effector functions through Fc region, while the latter include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). ADCP has been recognized one of the most important MoAs, especially for anti-cancer mAbs in recent years. However, traditional bioassays measuring ADCP always introduced primary macrophages and flow cytometry, which are difficult to handle and highly variable. In this study, we engineered a monoclonal Jurkat/NFAT/CD32a-FcεRIγ effector cell line that stably expresses CD32a-FcεRIγ chimeric receptor and NFAT-controlled luciferase. The corresponding mAb could bind with the membrane antigens on the target cells with its Fab fragment and CD32a-FcεRIγ on the effector cells with its Fc fragment, leading to the crosslinking of CD32a-FcεRIγ and the resultant expression of subsequent NFAT-controlled luciferase, which represents the bioactivity of ADCP based on the MoA of the mAb. With rituximab as the model mAb, Raji cells as the target cells, and Jurkat/NFAT/CD32a-FcεRIγ cells as the effector cells, we adopted the strategy of Design of Experiment (DoE) to optimize the bioassay. Then we fully validated the established bioassay according to ICH-Q2(R1), which proved the good assay performance characteristics of the bioassay, including specificity, accuracy, precision, linearity, stability and robustness. This RGA can be applied to evaluate the -ADCP bioactivity for anti-CD20 mAbs in lot release, stability testing as well as biosimilar comparability. The engineered cells may also potentially be used to evaluate the ADCP bioactivity of mAbs with other targets.