Ultrafiltration membranes of different pore sizes were used in enrichment of alpha-lactalbumin from whey solutions. Recovery and purity of the target protein was evaluated by different spectroscopic methods measuring total protein concentration and by RP-HPLC. The UV absorption, Bradford, Lowry and ninhydrin methods were tested with purified proteins present in milk and with skimmed milk and whey before they were applied to retention and fractionation analysis. The 30, 50 and 100 kg mol(-1) cut-off membranes appeared to be able to selectively fractionate alpha-lactalbumin to the permeate. Some of the spectroscopic methods were found to give very different responses for different proteins, and some methods measured also smaller protein based fragments. Furthermore, protein denaturation and aggregation, which can occur during the filtration process, increased the responses of the other methods except for the Lowry method. Hence, misleading membrane retention and fractionation results can be achieved if the method and standard protein used is not carefully chosen. The far UV and the Lowry methods appeared to be the most accurate and sensitive and had the highest precision for the main whey proteins. (C) 2011 Published by Elsevier B.V.