In the electrophoretic membrane contactor (EMC), a porous membrane is used to establish a contact across two flowing liquids between which an electrically driven mass transfer takes places. In this work, a methodology is proposed to select the best operating conditions to separate biomolecules in an EMC. Single-solution experiments were coupled with a theoretical approach to predict the influence of the process parameters (pH, membrane MWCO) on the separation factor. This methodology was applied to the separation of whey proteins, alpha-lactalbumin and beta-lactoglobulin, which are known to be difficult to separate. Experiments were first carried out with single synthetic protein solutions at different pH values (4.8, 6 and 8) using cellulose acetate membranes of either 30 or 100 kDa molecular weight cut-off. The experimental work was associated with a theoretical approach to study the mass transfer mechanisms. The parameters used in the model were calculated from the experimental variations of the solute and solvent transfer. The dependence of these parameters on the operating conditions gives the extent of electrostatic repulsion and provides information on the steric effect with respect to separation performance. The model was then used to calculate the separation factor for various operating conditions in order to determine the best ones (pH and membrane) for fractionation. Using the results, fractions enriched in alpha-lactalbumin and in beta-lactoglobulin were obtained at pH 4.8 with the 100 kDa membrane. (C) 2010 Elsevier B.V. All rights reserved.