A method for the detection and identification of penbutolol and its metabolites in human body fluid by using gas chromatography-mass spectrometry was established. The urine was extracted with diethyl ether-isopropanol and the plasma was extracted with Sep-Pak column. After TMS derivatization the sample was analyzed with GC-MSD. Six metabolites of penbutolol were found in urine sample. The recovery was 90.83% for urine and 85.88% for plasma. The detection limit of penbutolol was 5 pg.