This work expands the knowledge of the use of chemometric response surface methodology (RSM) in optimizing conditions for competitive binding partial filling ACE (PFACE), Specifically, RSM in the form of a Box-Behnken design was implemented in flow-through PFACE (FTPFACE) to effectively predict the significance of injection time, voltage, and neutral ligand (neutral arylsulfonamide) concentration, [L.], on protein-neutral ligand binding. Statistical analysis results were used to create a model for response surface prediction via contour and surface plots at a given maximum response (Delta RMTR) to reach a targeted K-b = 2.50 x 10(6) M-1. The adequacy of the model was then validated by experimental runs at the optimal predicted solution (injection time = 2.3 min, voltage = 11.6 W, [L-0 = 1.4 mu M). The achieved results greatly extend the usefulness of chemometrics in ACE and provide a valuable statistical tool for the study of other receptor-ligand combinations.