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The antibody fragments generated from hyperimmune equine IgG is widely used as anti-snake venom, anti-scorpion venom, anti-diphtheria, anti-tetanus, anti-gangrene and anti-rabies agents. Antibody fragments, F(ab) 2 , because of their specificity and absence of undesired reactivity are preferred over complete IgG. This paper discusses a novel purification technique for chromatographic purification of anti-rabies immunoglobulin G (IgG) fragment F(ab) 2 from horse serum. F(ab) 2 was purified by two successive chromatography steps using Cellufine A-200 and ProSep-vA Ultra media. The purified F(ab) 2 was characterized using biochemical and biophysical methods and shown to be pure and homogeneous. The purified F(ab) 2 was reactive to rabies antigen in immuno-electrophoresis and diffusion tests. The purified F(ab) 2 was biologically functional and was found to show a potency of 1500 IU ml −1 . Comparative analysis of the purity with commercially available F(ab) 2 by HPLC analysis and SDS–PAGE indicated that the present product is better in purity. To our knowledge, this is the first report providing evidence on purification of equine antibody fragment using controlled pore glass based protein A chromatography media.