A new high-performance liquid chromatographic (HPLC) assay suitable for pharmacokinetic studies of enantiomers of fenoprofen (FEN) was developed. Following the addition of internal standard (IS; racemic ketoprofen), the plasma or urine constituents are extracted into a mixture of isooctane:isopropanol (95:5), back extracted into water, and finally, extracted into chloroform. After evaporation of the organic layer, the drug and IS are derivatized with l-leucinamide hydrochloride via ethyl chloroformate intermediate. The formed diastereoisomers are chromatographed on a reversed-phase HPLC with a mobile phase consisting of monopotassium phosphate solution:acetonitrile:triethylamine (65:35:0.02) at a flow rate of 1 ml/min. The detection UV wavelengths are 232 and 275 for the drug and IS, respectively. The suitability of the assay for pharmacokinetic analysis of FEN enantiomers was examined by analysis of the plasma and urine samples taken from a healthy subject, following peroral administration of a single 300-mg dose of racemic FEN.