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A liquid chromatographic electrospray ionization mass spectrometric technique was developed for direct quantitation of iophenoxic acid (IA) in serum. IA was spiked into canine, feline, bovine, equine, and porcine sera, extracted, and quantified using negative ion monitoring following chromatographic separation on a Luna C18(2) 3 μm (100mm×2.1 mm) reversed-phase column. The limit of detection was 25 ng/mL and the limit of quantification was 50 ng/mL. Inter- and intra-assay accuracy (86–113% and 87–115%, respectively) and precision (1.8–7.7%) were calculated. Analysis of serum collected from feral pigs, raccoons, and opossums following ingestion of IA-marked baits confirmed the appropriateness of this method for bait acceptance studies.