The aims of this study were to devise a process for raising antibodies against Brazilian Bothrops venom in chicken egg yolks, to determine the best delipidation method for the preparation of the aqueous extract and to define the best purification conditions for IgY bothropic antivenom produced in eggs from hens immunized with Brazilian standard bothropic antigen. A group of nine Single Comb White Leghorn laying hens were immunized with venom from five different species of pit vipers of the genus Bothrops. The immunization process was carried out in three cycles, each performed six weeks apart. For extraction, the egg yolk was diluted 1:10 in distilled water, adjusted to a pH of 5.0, subjected to a freeze-thaw cycle, centrifuged and filtered before being precipitated with 20%(w/v) ammonium sulfate salt. This methodology retrieved 2.57 mg of IgY/ml of yolk from eggs. This preparation yielded antibodies capable of neutralizing lethal toxic activity of the pool of Bothrops sp venoms from five species, with an effective dose (ED50) of 365 μL/2 LD50 and, 1.0 mL of IgY antivenom neutralized 0.154 mg of venom. © 2009 Elsevier Ltd.