An enzyme-linked immunosorbent assay (ELISA) for paraquat is described. The microtitration plate-based assay has a limit of detection of 10 pg per well, and was specific for paraquat and monoquat. Reversed affinity chromatography was used to refine the polyclonal antibody preparation and eliminate interfering antibodies. There were consequent and significant improvements in assay sensitivity and performance. The potential for application of the assay to a variety of matrices is discussed.