Size-exclusion chromatography (SEC) was applied to purify cheese whey proteins α-lactalbumin and β-lactoglobulin which were present, respectively, in the polymeric and saline phases of aqueous two-phase systems. The optimal operating conditions for SEC using a HR 10/10® column packed with Sephadex G-25® medium led to a high degree of protein purification, of 99.7% for the saline phase and 99.6% for the polymeric phase. The values of resolution and process productivity were, respectively, of 1.53 and 0.43 mg/(mL h) for the saline phase and 1.29 and 0.31 mg/(mL h) for the polymeric phase. A general rate model predicted successfully the whey protein separation using a preparative SEC column. © 2003 Elsevier Ltd. All rights reserved.