beta-Lactamases are enzymes responsible for the hydrolysis of beta-lactam antibiotics, being produced by several pathogenic bacteria. Clavulanic acid is a commercially and clinically important beta-lactamase inhibitor, its extraction being possible by the application of aqueous two-phase system. In this study, clavulanic acid stability was investigated at different molar mass PEG (400, 1 000 and 20000 g mol(-1)) and at different citrate concentrations (5 and 20%) PEG/citrate aqueous-two phase systems (ATPS), under different pH values (4.0-8.0). Clavulanic acid extraction was also evaluated. Low citrate concentration and PEG 20000 (g mol(-1)) ATPS, at pH 6.0, were shown to be the systems that presented the highest clavulanic acid stability. Based on this, a factorial design 2(2) was used to evaluate CA extraction, being PEG (20,25 and 30%) and citrate (5, 10 and 15%) concentrations the parameters evaluated. Clavulanic acid was extracted into the PEG-rich phase (133.66 mg L-1), the highest values of the partition coefficient and yield being K = 5.92, Y = 103.53%, respectively The ATPS was not only effective on clavulanic acid extraction, but also its degradation was minimal. These results clearly indicate that ATPS can be successfully applied as a first step for the purification of clavulanic acid. (C) 2014 Elsevier B.V. All rights reserved.