A CE-based method for hexokinase inhibitor screening was developed in the present paper, in this method, hexokinase activity was assayed via electrophoretically mediated microanalysis (EMMA), which combines on-column hexokinase-mediated reaction and measurement of produced adenosine 5'-diphosphate (ADP) via electrophoretical separation and UV detection. Enzyme inhibition can be read out directly from the reduced peak area of ADP in comparison with a reference electropherogram obtained in the absence of any inhibitor. Conditions for on-column enzyme reaction and separation of adenosine 5'-triphosphate (ATP) and ADP were optimized. The optimal buffer composition for enzymatic reaction was 25 mM HEPFS buffer (pH 7.5) containing 5 mM MgCl2, whereas the optimal buffer composition for separation was 100 in M Tris-phosphate buffer (pH 5.5) containing 0.02% (m/v) hexadimethrine bromide (HDB). Fortunately, discontinuous buffer system call be adapted easily in the EMMA method. The time for separation was reduced dramatically to less than 3 min by reversing the direction of EOF via dynamically coating the capillary wall with the cationic polyelectrolyte HDB. Moreover, the peak tailing of ATP was also reduced by HDB coating. The Z' factor as high as 0.98 was obtained, indicating a high quality of the screening data. The present method is simple, robust and cost-effective.