In this work, nonionic biocompatible Tween 20 based aqueous two-phase systems (ATPSs) were studied. These systems provide a gentle media with low interfacial tension for extraction of biomolecules. In accordance with the results, the top phase tends to be surfactant-rich and the bottom phase tends to be salt-rich. Binary and ternary phase diagrams were plotted and their trends were correlated with the effective excluded volume (EEV) parameter and Setschenow equation. Also, experimental binodal data were successfully fitted to Merchuk correlation. Moreover, after plotting tie lines, their consistency was analyzed through applying the Othmer-Tobias and Bancroft correlations. Afterwards, tie line length (TLL) and slop of tie line (STL) were calculated. Furthermore, cefalexin partition coefficients along with yield recovery in three different temperatures were determined and the influence of hydrogen bonding and salting-out effect were studied. Finally, role of Hofmeister series and effect of salts being applied in these systems on cefalexin partition coefficient in each phase, were investigated.(C) 2014 Elsevier B.V. All rights reserved.