In this study, three phase partitioning (TPP) as an efficient bioseparation method, was explored for the first time to purify stable uncontaminated L-asparaginase from Capsicum annuum L, and its antitumor activity was evaluated. With the optimized system parameters, the enzyme was purified to homogeneity and having 6.83-fold with 567.4% recovery of its activity. The optimum pH and temperature of the TPP purified enzyme were determined as 8.5 and 40 degrees C, respectively. The stability studies of the enzyme activity envisaged that the enzyme is stable up to 45 degrees C and retained its activity over a wide range of pH (5.0-9.0). The purified enzyme showed good scavenging activity, and its total antioxidant capacity was found to be 88.58 mu M ascorbic acid equivalent. The antiproliferative activity of the purified L-asparaginase was also investigated against the three human cancerous cell lines. (C) 2015 Elsevier B.V. All rights reserved.