The partition coefficient of proteins (carbonic anhydrase from bovine (CAB) and lysozyme treated under reducing conditions) was quantitatively evaluated in Breox/dextran aqueous two-phase systems in order to develop a stress-mediated bioseparation process using the conformational change of proteins and the stimuli-responsive aqueous two-phase systems. A comparison of the partition coefficients with the surface properties, previously characterized, shows that the change of protein conformation and their surface properties (i. e. surface net and local hydrophobicity) can be estimated simply by using the partition coefficient and without analyzing of the properties of the partitioning systems. The values of both the stimuli (temperature and GuHCl concentration) and the conformational change, estimated from the partition coefficients, were normalized As a result, the combined stimuli of temperature and GuHCl concentration to determine the protein conformation were found to be expressed as a linear summation of them. The methodology for the design of the separation process was investigated based on the diagram for GuHCl concentration and temperature, in which the local hydrophobicity was maximal, and the separation of lysozyme and CAB was achieved as a model case of the methodology.