The whey protein separation potential of aqueous two-phase systems of arabinogalactan [AG] (Lonza FiberAidTM) and polyethylene glycol [PEG], buffered with 10 mmol/g phosphate or citrate buffer, was studied. 100 mmol/g potassium chloride [KCl] was added as required. Previously-published phase equilibrium results were verified and the absorbance of whey protein isolate [WPI] in an AG-PEG solution was measured. The effect of pH, KCl concentration, initial WPI concentrations and upper to lower phase mass ratios on whey partitioning was studied. The best separation system contained 17.20% (w/w) AG, 7.20% (w/w) PEG, 10 mmol citrate buffer (pH 5.4) and 100 mmol KCl per gram of total system. The upper to lower phase mass and volume ratios were 1:1 and 16:11 respectively. Approximately 12 mg (mainly α-lactalbumin) of the 20 mg WPI added partitioned into the AG-rich upper phase. This system has potential to reduce chromatographic requirements in large scale separation of protein mixtures.