A direct sandwich enzyme-linked immunosorbent assay (ELISA), employing affinity purified antivenom antibodies specifically recognizing the homologous venom, was developed for species-specific detection of bothropic venom. the method is based on a two-step affinity purification of the specific antibodies. A species monovalent antivenom is adsorbed onto a venom adsorbent containing heterologous venoms from theBothrops, CrotalusandLachesisgenera. the species-specific antibodies obtained, are then adsorbed onto a second venom adsorbent containing only the homologous venom for the removal of non antivenom antibodies. Venom concentrations of 0.1 and 1,000 ng/ml were specifically identified forBothrops jararacussuandB. alternatusvenom respectively.