The only definitive management of snake envenoming is the use of snake antivenom. Endotoxin contamination is a serious threat to the safe use of parenteral drugs. A greater understanding of the nature of limulus amebocyte lysate (LAL) test interference and use of permissible dilutions has minimized enhancement problems. Common interference issues include suboptimal pH, enzyme or protein modification, and nonspecific LAL activation. This study aimed at determining the interference factors associated with validating the antivenom sera preparations to avoid false-positive results when testing snake antivenom serum samples by the LAL method. Phase I (preliminary screening/interference assay) was performed to determine a compatible test dilution, which was then used in Phase II (inhibition-enhancement/validation study). The best approach to resolve interference issues was dilution by 1:80 (maximum valid dilution) plus a specific treatment as heat-activation at 70°C-80°C for 10 min with rehydration of LAL reagent with endotoxin-specific buffer solution.LAY ABSTRACT: Snake antivenom sera are produced by immunizing horses with repeated nonlethal doses of snake venom. Bacterial endotoxins constitute one of the major problems in the formulation of pharmaceutical products. One such method for detecting endotoxin levels is the bacterial endotoxin test (BET). However, some substances show strong interfering action with the BET that cannot be avoided by simply diluting the sample solution. In this work, the test for interfering factors was performed as two identical series of product dilutions-one spiked with 2λ and one left unspiked. The result of the interference test revealed the noninterfering dilution (NID) of the product, which was used for the actual validation. Our results showed that after treating the samples using different procedures, such as heat activation at 70-80°C for 10 min followed by centrifugation at 2000 rpm for 10 min and dilution of samples in BD100 (biodispersing agent), inhibition and enhancement up to 1:100 maximum valid dilution (MVD) were observed. Finally, to resolve this inhibition/enhancement problem, the activated sample was heated at 70-80°C for 10 min with rehydration of the Endosafe LAL reagent in an endotoxin-specific buffer solution (BG120) to block β-d-glucans and limulus amebocyte lysate (LAL) reactive material (LAL-RM).