Objective To establish a reliable platform for screening glucokinase activators (GKAs) in vitro. Methods Pancreatic glucokinase (PGK) protein expressed in a prokaryotic expression system as a histidine-tagged fusion protein from Homo sapiens was produced. Then, response surface methodology (RSM) was used to optimize the microplate-based GKA screening platform. In the first step of optimization with Plackett-Burman design (PBD), initial pH, reaction time and MgC12were found to be important factors affecting the activity ratio of GKA (RO-28-1675) significantly. In the second step, a 23 full factorial central composite design (CCD) and RSM were applied to the optimal condition determination of each significant variable. A second-order polynomial was determined by a multiple regression analysis of the experimental data. Results The following optimal values for the critical factors were obtained: initial pH 0 (7.0), reaction time-0.63 (13.7 min) and MgCl2 0.11 (2.11mmol/L) with a predicted value of the maximum activity ratio of 34.1%. Conclusion Under the optimal conditions, the practical activity ratio is 34.8%. The determination coefficient (R2) is 0.9442, ensuring adequate credibility of the model.LLAE3, extracted from Folium nelumbinis in our laboratory, has prominently activated effects on PGK.