A new quantitative and selective assay was developed for the measurement of low levels of clonidine in human plasma. The drug and the deuterated internal standard (2H4)clonidine are quantified by gas chromatography/negative ion chemical ionization mass spectrometry with methane as the reagent gas. After a three-step extraction procedure, the two compounds of interest are converted to their fluorinated derivatives using 3,5-bis(trifluoromethyl)benzoyl chloride. The mass spectrometer is focused to monitor abundant and stable characteristic high-mass ions (m/z 673 and 709) which are generated in the ion source by a resonance electron capture process. This assay requires only 1 ml of plasma sample and the limit of detection of the method is 5 pg ml-1 with a relative standard deviation of less than 7%.