In single stage operation, high quality separation of similar size proteins was performed via batch ultrafiltration. Two permeable proteins, hemoglobin and ovalbumin, were separated using a 100 kDa polyethersulfone membrane for a volume concentration factor of 3.5. At low ionic strength, hemoglobin and ovalbumin showed their enhanced reverse selectivity of 11 and selectivity of 14 at pH 9 and 3.5, respectively. Protein adsorption on the membrane during ultrafiltration was carefully controlled by fine adjustment of pH and ionic strength of feed. The high separation factor was achieved as a result of pore constriction by protein adsorption and the electrostatic interaction between proteins and the membrane.