Imidazolium oximes are useful in the treatment of organophosphorus agent poisoning. However, the extant methods for analyzing oximes in plasma and urine samples are not adequate. A unique high-performance liquid chromatographic assay method was developed for quantitating either imidazolium or pyridinium oximes. Plasma or urine samples can be injected directly onto the column after a centrifugation filtration step. This method demonstrates a different approach in the method development for quaternary ammonium compounds using non-end-capped reversed-phase columns and a mobile phase containing competing cations. In addition, a preliminary pharmacokinetic study of the imidazolium oxime in rabbits was carried out using this method.