Control of external pH and ionic strength is used to separate proteins with surface-modified, nanoporous polycarbonate track etched (PCTE) membranes. The porous PCTE membranes were modified with monolayers of self-assembled thiols (HSC10H20COOH) on electroless gold. The hydraulic radius of the pores in the surface-modified membranes was 8.7 nm. Two proteins of nearly identical molecular weight, bovine serum albumin (BSA) and bovine hemoglobin (BHb), were used as the permeants. The fluxes of BSA and BHb through the membranes show maximum values at the isoelectric points (pI) of the proteins. At pH values above and below the pI, charge interactions between the proteins, their counterions, and the pore surface leads to a decrease in flux. The imposition of a difference in ionic strength across the membrane causes osmotic flow and leads to a significant increase in the protein fluxes and an enhancement of the selectivity of BSA over BHb. In protein separation experiments, the BSA and BHb fluxes are nearly 3 times larger than those observed with no ionic strength difference.