Ultrafiltration (UF) separation of hemoglobin (Hb, pI 7.1, MW 68,000) and bovine serum albumin (BSA, pI 4.7, MW 67,000) in aqueous Solutions With PES (polyethersulfone, MWCO = 100 kDa) and PAN (polyacrylonitrile, MWCO = 100 kDa) membranes was studied. Experiments were performed by changing operating parameters including Solution pH (4-7.5), initial protein concentration (100-500 ppm), transmembrane pressure (TMP, 10-50 psi), ionic strength (0.01-0.1 M), and stirring speed (100-300 rpm). More effective separation was achieved at a lower protein concentration, I lower TMP, or a pH above the pI of Hb with PAN membrane. Under the conditions of low pressure, low protein concentration, and low ionic strength, the interactions between the charges of proteins and membranes were important. The flux decreased more sharply when pH was lower than 7.1. Response surface methodology by the Box-Behnken model was used to examine the role of each process variable on protein rejection and UF flux. A second-order polynomial regression model could properly interpret the experimental data with an R2 value of 0.97, based on the estimated rejection of BSA LIP to 99.7%. Interactions between these operating parameters and the significance effects on UF operation were also discussed.