A competitive enzyme-linked immunoassay (ELISA) for the quantitation of Penoxsulam [2-(2,2-difluoroethoxy)-6-(trifluoromethyl-N-(5,8-dimethoxy[1,2,4]triazolo[1,5-c]pyrimidin-2-yl))benzenesulfonamide] in ground and surface waters was developed. This immunoassay utilizes magnetic particles as the solid phase to which polyclonal rabbit anti-Penoxsulam antibodies are attached. The ELISA has an estimated detection limit of 0.17 ppb (microg/mL) of Penoxsulam in water. Specificity studies indicate that the antibody can distinguish Penoxsulam from its major metabolites and structurally similar pesticides. Interference studies indicate that the ELISA has a wide tolerance of sample pH and salinity and for compounds commonly found in surface and ground waters. The ELISA was shown to compare favorably to LC-MS/MS on ground and surface water samples (r(2) = 0.957). The various studies performed demonstrate the usefulness of the ELISA technique as a rapid and high-throughput analytical method for the cost-effective monitoring of water samples.