A kinetic enzyme-linked immunosorbent assay (ELISA) was developed using antigenic extracts prepared from Psoroptes cuniculi mites and sera from 37 Psoroptes sp.-infested and 43 uninfested bighorn sheep (Ovis canadensis). Serial dilutions of these serum samples, representing 3 bighorn sheep subspecies and 9 geographic areas, gave parallel responses when plotted as log dilution versus log kinetic rate. Therefore, all 80 samples were run at a single dilution (1:100) and positive/negative cutoff values were established as the mean kinetic rate of all negative sera plus either 2, 3, or 4 standard deviations. The resulting ELISA was highly reproducible and accurate with sensitivities and specificities of 100% and 97.7%, 94.6% and 97.7%, and 94.6% and 100%, respectively. This immunoassay will be useful for prospective and retrospective studies assessing the distribution and prevalence of Psoroptes sp. infestations in bighorn sheep.