A new and sensitive gas chromatographic-mass spectrometric (GC-MS) assay for routine analysis of clonidine in human plasma is described. Quantification is carried out with simple electron-impact ionization mass spectrometry and solid-phase cartridges for preliminary extraction from plasma. The pentafluorobenzyl derivative of clonidine yields an intense ion fragment at m/z 354, and the lower limit of detection is 0.025 ng/ml for a 1-ml plasma sample. The practical applicability of this method is demonstrated by determining plasma concentrations of clonidine in a clinical study of a new transdermal delivery system for clonidine.