Asymmetric membranes based on the interfacial cross-linking of poly(vinyl alcohol) hydrogels with toluene diisocyanate exhibit enhanced size selectivity for binary protein separations with minimal reductions in the flux of the smaller protein. The asymmetric nature of the hydrogel prevents the use of swelling measurements to characterize the mesh size of the selective surface layer. Alternatively, the permeability coefficient of the surface layer and the contribution of this layer to the overall resistance were estimated. A direct relationship was found between the surface layer permeability coefficient and the amount of urea/urethane groups, determined from infrared spectroscopy, introduced into the surface during interfacial cross-linking. Impregnation of the hydrogel within the pores of a microfiltration membrane for mechanical support was demonstrated and the transport properties of this composite membrane compared well to those of an unsupported hydrogel.