Two selective and sensitive methods for the quantitative analysis of butofilol in human plasma and urine are discussed. The first method is a gas chromatographic assay with electron-capture detection using extraction with toluene, several clean-up procedures and derivatization. The second method is based on high-performance liquid chromatography and a single extraction with dichloromethane. The two assay methods were applied to the determination of the same human plasma samples after administration of a single, oral 200-mg dose of butofilolol. A good correlation between the results (inter-laboratory comparison) is obtained, validating both techniques.