This paper describes the validation of an anal. method previously developed to measure in BALB/c mice, the neutralizing potency of an anti-Loxosceles plasma against recombinant toxins of medical relevant Loxosceles species. To the method validation, different parameters defined in the International Conference of Harmonization guidance including accuracy, precision, inter-assay variation, reproducibility, specificity and selectivity were evaluated. The method is specific, selective, accurate and precise with variation coefficient less than 20 % and its reproducibility interassay was demonstrated between different days and inter-analysts. This validation demonstrated that the method is statistically suitable and may be used as quality control test by manufacturers as part of plasma release for the production of the anti-Loxosceles antivenom.