In recent decades, the use of aqueous two-phase system (ATPS) for bio-separation engineering has attracted increasing interest. In this work, ATPSs based on carbohydrates and acetonitrile were used to explore the partitioning behavior of cephalosporin antibiotics including cephalexin, cefazolin, and cefixime. For this purpose, four ATPSs containing acetonitrile and glucose, fructose, sucrose, and maltose have been studied. Initially, the phase diagrams were acquired at 300 K and then correlated by the Merchuk equation. The results showed that the ATPS composed of disaccharides have a broader two-phase region than monosaccharides. Furthermore, for the studied systems, tie-lines were obtained and their accuracy was examined by Othmer-Tobias and Bancroft equations. Moreover, tie-lines length and slope of tie-lines were determined. Besides, the partition coefficient of cephalosporins in four different ATPSs and a total of 48 tie-lines were investigated. Based on the results, it can be concluded that by increasing the concentration of carbohydrates, the partition coefficient increases. The maximum partitioning coefficient of cephalexin and cefazolin were obtained to be 19.2 and 17, respectively, with a recovery yield of 93% in the carbohydrate phase, which corresponds to the system (16 wt % glucose + 50 wt % acetonitrile + water). While the highest partitioning coefficient of cefixime was 6.4, with a recovery percentage of 81% for the system (16 wt % sucrose + 50 wt % acetonitrile + water). The results of this work indicate that these systems have a potential for separation and recovery of the understudied antibiotics. (C) 2019 Elsevier B.V. All rights reserved.