Abstract Recently developed aqueous two-phase systems based on non-ionic detergents and polymers are suitable for the separation of membrane proteins. Moreover, within this relatively membrane protein “friendly” environment, changes in temperature can be controlled and stabilizing agents may be added to ensure integrity of the target protein during isolation. Here, we use aqueous two-phase partitioning for the isolation of membrane bound 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). Different detergents were used to find optimal conditions regarding solubilization and retaining target protein activity. We explored in situ solubilization by adding detergent directly to the aqueous two-phase system, as well as a batch metal affinity capture step of 6xHis tagged 11β-HSD1 in the two-phase system. The use of detergent/polymer two-phase systems resulted in a specific enzyme activity of 3840 nmol mg −1 min −1 of the target membrane protein compared to a conventional purification protocol where a specific enzyme activity of 1440 nmol mg −1 min −1 was achieved.