BACKGROUND: The high potential toxicity of epsilon toxin (Etx) produced by Clostridium perfringens (C. perfringens) type D, has made it the third most lethal clostridial toxin behind botulinum and tetanus, therefore, having a pure and concentrated Etx is very important. OBJECTIVES: The aim of this study was to purify Etx as pure as possible with an applicable, cost-effective, multistep purification protocol with the lowest and shortest time. METHODS: The purification of the Etx was carried out in multiple consecutive steps; ammonium sulfate precipi-tation, dialysis, size exclusion chromatography by G50, two concentration steps, and ultrafiltration. The Etx activity after different steps was evaluated by the minimum lethal dose (MLD) calculation, according to the standard oper-ating procedure. Toxin quantification was determined using Lowry technique, and its presence and specificity was tracked to identify pure Etx by SDS- PAGE and western blotting. Finally, the purity of Etx was evaluated by capil-lary electrophoresis. RESULTS: The purified Etx formed a single band of about 32.9 kDa in SDS-PAGE and blotting. The pure Etx concentration was calculated to be 3.9 mg/ml and its MLD value was the dilution of 1/24000 after the ultrafiltration step. The presented purification processes to purify Etx resulted in ~ 87-fold concentration and 88.6% purity. CONCLUSIONS: Due to this high Etx purity, the processes used in this study can provide the technical knowledge of toxin production in a larger industrial scale that can be used in development of clostridial toxoid vaccines, as well as quality control and/or diagnostic tests.