Aqueous two-phase partition and triphasic systems containing thiophilic and metal ligands were used to produce anti-snake venom serum using the fractionation of hyperimmune equine plasma with neutralizing activity against Bothrops spp. venom as the model. In poly (ethyleneglycol) (PEG)/salt systems, PEG molecular weight, system pH, and the amount of polyvinylpirrolidone (PVP) added were optimized in order to promote selective immunoglobulin recovery. From these studies a PEG 1500/ammonium sulfate system, pH 8.0, a tie-line length parameter (TLL) 21%, and a 2% PVP K-12 were preliminarily selected. The immunoreactive fraction (ELISA) mainly preferred the top phase; with a theoretical yield of > 97% and a purification factor of 3.5. Practical recoveries were, however, hampered by limited product solubility and an erratic global process performance. In order to overcome these limitations, we moved to a system formed by derivatives of cellulose and starch, which contained an affinity solid phase. This three-phase system was compatible with the use of thiophilic or immobilized metal ion affinity (IMAC) supports, the first showing the higher selectivity. The system composed of 2% Methocel, 2% Reppal, 1.0 mol/kg ammonium sulfate, and 5-10% 2S-thioether sulfone agarose allowed recovery of 52% of the immunoreactive fraction from hyperimmune equine plasma at a total protein concentration of 0.25%. This process strategy also allowed simple operation and easy of affinity ligand recycling.