An easy, sensitive, competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) for spectinomycin in chicken plasma was developed. Preparation of a spectinomycin-bovine serum albumin conjugate in which the hapten is linked to the carrier protein through the C-4 position is described. Antibodies raised against antigens in rabbits had excellent specificity for spectinomycin, exhibiting a cross-reactivity of 44.0% with dihydrospectinomycin and 13.8% with tetrahydrospectinomycin. No cross-reactivity was observed with other antibiotics. The detection limit of the CI-ELISA was 2 ng/mL (equivalent into 40 ng/mL undiluted chicken plasma) spectinomycin. Known amounts (0.1-100 micrograms/mL) of spectinomycin were added to chicken plasma and then analyzed. Average recoveries were 97-110%. This procedure may be used without prior extraction of samples.