Abstract: The biological activity of ADCC by anti-CD20 monoclonal antibody was determined by BioGlo^TM Luciferase Assay System using Jurkat/NFAT-luc+Fc γRIIIa cell line as effector cell and WIL2-S cell line as target cell. The developed method was verified for specificity, precision and accuracy. Anti-CD20 monoclonal antibody showed a dose-response mode by the developed method, and the determination result complied with the following four-parameter equation: y=(A-D)/1+(X/ C) ^B +D. The optimized parameters of the method were determined including the antibodies diluted concentration (18 000 ng·mL^-1), dilution rate (1:5), the ratio of effector cell and target cell (6:1), and induction time (6 h). The values of eight independent tests have passed a statistical test for curve regression analysis, linear or parallelism, which showed the method possessed good specificity. Four different dilute groups of recovery rates sample were determined for 3 times, and the result showed mean relative potencies of (44.39 ± 3.93)%, (72.74 ± 2.78)%, (128.28 ± 7.01)% and (168.19 ± 2.70)% respectively, with a variation coefficient of less than 10%, and the recoveries of (88.78 ± 7.85)%, (96.99 ± 3.70)%, (102.63 ± 5.61)% and (112.12 ± 1.80)% respectively. A novel reporter gene method for determination of biological activity of ADCC by anti-CD20 monoclonal antibody was successfully developed, which showed strong specificity, good reproducibility and high accuracy, and might be used routinely.