6-Aminopenicillanic acid (6-APA) is used for synthesis of semisynthetic antibiotics. Polymer-salt aqueous two-phase systems (ATPSs) were applied for separation of 6-aminopenicillanic acid (6-APA) and phenyl acetic acid (PAA), as the products of hydrolyzation reaction of Penicillin G / Penicillin V. The binodal curves of ATPS composed of a copolymer (reverse Pluronic 10R5, Pluronic L35 and PEG-ran-PPG) and a salt (Tri-sodium citrate, tri-potassium citrate, di-potassium phosphate, sodium sulphate and magnesium sulphate) were obtained. The results show that, at a fixed PPG/PEG ratio, block copolymers have larger two-phase region compared with random copolymer. After screening on the partition coefficient of PAA and 6-APA separately, Na2SO4 was selected for studying the effect of the copolymer structure and the composition of salt and copolymer on partitioning, considering higher selectivity of PAA and 6-APA. 10R5-Na2SO4 ATPS was selected as the most appropriate system for separation of 6-APA and PAA. This system was used for separation of mixture of 6-APA and PAA. The results show that selectivity was 53 and smaller in a system, containing a mixture of 6-APA and PAA. This observation can be justified by the interaction between 6-APA and PAA. Molecular interaction between these two molecules were investigated by the Flory-Huggins interaction parameter.