Every year large numbers of venomous snake bites occur around the world, especially in tropical areas. The World Health Organization classifies venomous snake bites as one of their highest priority neglected tropical diseases, one of the reasons being the short supply of antivenom compared to the number of snake envenomations. The standard of care for snake envenomation is administration of antivenom. Many antivenoms are polyvalent, which are produced using venoms from multiple species of snakes. The polyvalent antivenoms can treat envenomation from snake venoms used in the production, but also show cross-reactivity against snake venoms with similar composition. Determining cross-reactivities of antivenoms could help improve the quality of treatment and provide a better understanding of venom-antivenom binding. One antivenom only has been available in the United States for treatment of North American Crotaline envenomation, with the recent introduction of an F(ab’)2 antivenom (ANAVIP®). Size-exclusion high performance liquid chromatography (SE-HPLC) was used to assess cross-reactivity of the western pygmy rattlesnake, Sistrurus miliarius streckeri (S. m. streckeri), against ANAVIP®. Estimates of venom-antivenom reactivity was measured in reaction mixtures based on the increase in elution profile area of higher molecular weight complexes (region 1) and on the decrease in elution profile area of reactants (region 2). Reaction mixtures contained ANAVIP® (1.0 mg/ml) and S. m. streckeri venom (0.125, 0.25, 0.5, or 1.0 mg/ml). Controls were ANAVIP® and S. m. streckeri (1.0 mg/ml). Mixtures were incubated at 37 °C for 30 min, then stored at 4 °C (5 min) prior to SE-HPLC. Relative binding, estimated from the increase in region 1 (immune complexes) and decrease in region 2 (reactants) region areas, suggested saturation of reactive antivenom binding sites at 0.125 (and above) mg venom/mg antivenom. SE-HPLC data indicate that binding of ANAVIP® to S. m. streckeri venom does occur, consistent with protective effects observed clinically. Further studies are needed to compare the binding of S. m. streckeri venom to other commercially available antivenoms, and the binding of ANAVIP® to other venoms of clinical significance.