BACKGROUND: This work describes a flowsheet optimization framework for the development of protein purification operations involving aqueous two-phase protein extraction (ATPE). The framework encompasses both the flowsheet and thermodynamic calculations and predicts the optimal flowsheets for the application of ATPE systems. RESULTS: Results presented were generated by applying an optimization algorithm to a previous case study with demonstrated feasible solutions(1) concerning the recovery of phosphofructokinase from a simple model feedstock containing both phosphofructokinase and ovalbumin using poly(ethylene glycol)-dextran (PEG6000-DxT500) two-phase systems. An optimal flowsheet for a one-stage extraction unit and improved flowsheets for two-stage extraction units with and without recycle were determined based on the minimum scaled process cost subject to attainment of liquid-liquid equilibrium in the ATPE system(s) and constraints on product yield, product purity and phase settling rates. The cost performance factors of feasible one-stage and two-stage flowsheets with and without recycle were improved by 4.7%, 8.6% and 33.1%, respectively, relative to the prior work. CONCLUSION: This flowsheet optimization framework was able to predict the optimal flowsheets for protein purification from a two-protein mixture using ATPE systems, subject to some limitations. The algorithm has the potential to be successfully applied to predict protein extraction from complex mixtures upon improvement in its computational robustness. (C) 2012 Society of Chemical Industry