A sensitive and reproducible enzyme-linked immunosorbent assay (ELISA) for the determination of the concentration of ivermectin (IVM) in biological fluids was developed. A conjugate of IVM on bovine serum albumin and poly-l-lysine was used to produce antibodies in rabbits and served as a solid-phase marker for titration of antibodies, respectively. The competitive ELISA was conducted by simultaneously incubating IVM and IVM-biotin conjugate with anti-IVM antiserum over goat anti-rabbit IgG (Fc) and then determining the amount of bound IVM-biotin with avidin-peroxidase conjugate as a tracer. The coefficient of variation for the assay was less than 10% in the range of 0.3–10 ng/ml. The limit of detection was 0.1 ng/ml. The cross-reactivities of anti-IVM antiserum with some anthelmintic drugs were negligible. Using this ELISA, serum levels of IVM were easily determined in Mongolian jirds (Meriones unguiculatus) up to 72 hr following a single oral dose of 500 µg/kg of body weight.