A sensitive and selective method for the quantitative analysis of prenalterol in plasma and urine is described. Prenalterol and the internal standard, deuterated prenalterol, are extracted with diethyl ether at pH 9.9 under salting-out conditions. Derivatization is performed by means of pentafluoropropionic anhydride in toluene before separation in the gas chromatograph. Detection and quantification of the triacyl derivatives are done by mass spectrometry in the selected ion monitoring mode. The method allows determination of concentrations down to 5 nmol/l (1 ng/ml) in 1 ml of sample, with a relative standard deviation below 10%.