Although, previous studies have demonstrated the importance of electrostatic interactions in membrane systems, there are few techniques available for the accurate analysis of these effects. This manuscript describes a novel approach to study electrostatic interactions during ultrafiltration using protein charge ladders. which consist of a series of chemical derivatives of a given protein that differ by single charge units. Charge ladders were formed by reacting myoglobin and bovine carbonic anhydrase with acetic anhydride to block free lysine groups. The concentration and net electrical charge of the individual "rungs" were determined by capillary electrophoresis. Sieving experiments were performed using a simple stirred ultrafiltration cell. The results clearly show an increase in electrostatic exclusion of the more negatively-charged species in low ionic strength solutions. The membrane surface charge density was estimated from myoglobin sieving data by comparing the results to model calculations. This surface charge density was in good agreement with independent measurements of the membrane zeta-potential. These results demonstrate that protein charge ladders can be used to study electrostatic interactions in much the same way as polydisperse dextrans have been used to study steric interactions in membrane systems.