A sensitive and stereoselective high-performance liquid chromatographic assay for the quantitative determination of the analgesic tramadol and Odemethyltramadol, an active metabolite, is described in this work. Ketamine was used as internal standard. The assay involved a single tert-butymethylether extraction and liquid chromatography analysis with fluorescence detection. Chromatography was performed at 20ยฐC on a Chiracel OD-R column containing cellulose tris-(3,5-dimethylphenylcarbamate) as stationary phase, preceded by an achiral end-capped C 18 column. The mobile phase was a mixture of phosphate buffer (containing sodium perchlorate (0.2M) and triethylamine (0.09M) adjusted to pH 6) and acetonitrile (80:20). The method developed was validated. The limit of quantitation of each enantiomer of tramadol and its active metabolite by this method was 0.5 ng/mL; only 0.5 mL of the plasma sample was required for the determination. The calibration curve was linear from 0.5 to 750 ng/mL for tramadol enantiomers, and from 0.5 to 500 ng/mL for O-demethyltramadol enantiomers. Intra and interday precision [coefficient of variation (CV)] did not exceed 10%. Mean recoveries of 95.95 and 97.87% for (+)R,R-and (-)S,S-tramadol and 97.70 and 98.79% for (+)R,R-and (-)S,S-O-demethyltramadol with CVs <2.15% were obtained. Applicability of the method was demonstrated by a pharmacokinetic study in normal volunteers who received 100 mg of tramadol by the intravenous route.