To reduce the electrical energy consumed in electrokinetic separation processes, a stacked membrane system composed of two polypyrrole (PPy)-coated porous membranes and a microporous polycarbonate filter (PCTE) has been suggested. In this PPy/PCTE/PPy membrane system, the outer two PPy membranes provide working electrodes for the migration of ions through the PCTE pores. Since the spacing between the two PPy membrane electrodes is only the thickness of the PCTE membrane, a strong electric field can be obtained by a small potential difference between the electrodes. Using this electrokinetic separation system, positively charged L-lysine (Lys) and negatively charged L-aspartic acid (Asp) are separated from their binary mixture at pH 4.7. For the efficient separation of both ions, the operation is carried out in a continuous and cyclic mode. The effects of potential difference on separation performance are investigated by varying the electric potential from +/-1 to +/-5 V. A higher potential difference results in a better separation. The key parameters determining the optimal experimental conditions are the electrokinetic migration and the pressure-driven convection rate of ions.