A high performance liquid chromatographic (HPLC) assay was developed for the determination of pimethixene in plasma and urine. Pimethixene was extracted by a rapid and simple liquid-liquid procedure using n-pentane:isopropyl alcohol (50:1), and a reversed phase HPLC separation technique with UV spectrophotometric detection at 254 nm. Determination was possible for pimethixene in the concentration range 53–530 ng ml with the limit of detection at 20 ng/ml for both plasma and urine. The overall recoveries of pimethixene added to plasma and urine samples by an external standard method were 95.6 and 98.5%, with coefficients of variation of 0.9993 and 0.9997, respectively.