AbstractSeparation of bovine serum albumin (BSA) and hemoglobin (Hb), two proteins with almost identical molecular weight, has been a big challenge for more than two decades. Using traditional ultrafiltraion membranes separation of these proteins is possible only at the isoelectric point of one protein via electrostatic repulsion between the membrane and the other protein. Here we introduce an integral asymmetric isoporous membrane from polystyrene-block-poly(2-hydroxyethyl methacrylate-ran-2-(succinyloxy)ethyl methacrylate) (PS-b-P(HEMA-r-SEMA)) having random –OH and –COOH groups along the pore walls prepared by a method which combines solvent induced self-assembly of the block copolymer and nonsolvent induced phase separation (SNIPS). The membrane consists of soft isoporous channels due to swelling of the P(HEMA-r-SEMA) blocks in a hydrated state. The effective pore size of the membrane in a hydrated state is significantly lower compared to that in a dry state. These soft channels allow the permeation of BSA while retaining Hb at constant pH 7.4 where both proteins are negatively charged. In comparison to 22 commercial and in-house prepared membranes the PS-b-P(HEMA-r-SEMA) membrane presents unprecedented ideal selectivity of BSA over Hb (). Unlike the conventional technique in this case the electroneutrality of one protein is not mandatory which will provide significantly easier handling.