A novel assay procedure for ketamine, based upon the extraction of xylene red B into an organic solvent in direct proportion to the amount of ketamine present, is described. Dye concentration is measured by fluorescence, permitting assays in the 0–1 μg range. Some interference by the N-dealkylated metabolite of ketamine was encountered, and other basic drugs may interfere with the assay. With certain limitations, the fluorescent dye procedure appears suitable for clinical application.