An unsteady state mathematical model describing the continuous separation process of a binary mixture of protein using partial recycling free-flow isoelectric focusing is constructed. The model consists of a one-dimensional mass balance equation and an electric charge balance equation for the electrophoretic chamber and a mass balance equation for the reservoir. The model equations were solved numerically using a finite difference method with literature values of physical constants such as dissociation and diffusion constants. The calculated results are in good agreement with the experimental data for the separation of protein mixture of bovine serum albumin (BSA) and bovine hemoglobin (Hb) reported previously. Furthermore, the model can describe the effects of the operating factors, such as support electrolyte concentration and feed flow rate, on the separation process.