The pharmaceutical and food industries are interested in capsaicin because of its therapeutic and functional potential; however, it is necessary to develop an efficient protocol for extraction and purification of capsaicin from natural sources. Thus, this paper aims to study the partitioning and purification of capsaicin from capsicum peppers using aqueous two-phase systems (ATPSs) based on Na+ or Kt. salts. Initially, phase diagrams were determined, and the ease of phase formation of acetonitrile + different salts ATPS followed the reverse of Gibbs free energy of hydrations. Capsaicin migrated preferentially to the top phase (acetonitrile-rich phase) by an endothermic and spontaneous process governed by entropic forces. The best condition of separation (K-CPS = 43.95 +/- 1.52; EECPS = 89.06 +/- 1.17%; PFCPS = 2.65 +/- 0.01 fold) was found using acetonitrile (20 wt %) + K2CO3 (15 wt %) + water (65 wt %) at 318 K and 0.1 MPa.