The partitioning behavior of glutaminase produced from Zygosaccharomyces rouxii in polyethylene glycol (PEG)-salt aqueous two-phase systems (ATPSs) was investigated. ATPSs comprising of different PEG-salts were considered. Binodal data and tie lines generated for the selected systems were analyzed and correlated with Othmer-Tobias and Bancroft equations. Effects of salt type, PEG molecular weight, concentrations of phase components, and tie line length on enzyme partitioning were evaluated.