A procedure for the determination of the CNS-active muscle-relaxant mephenesin in plasma samples is described. The method involved a single-step extraction with diethyl ether followed by high-performance liquid chromatography separation and fluorimetric detection. Linearity of the detection response was observed between 1 ng/ml, which is the limit of determination, and 500 ng/ml. Inter-day assays, including quality-control samples, performed over a 4-month period, showed the method to be reproducible and precise, and suitable for routine analysis in pharmacokinetic studies.