We report an isotope dilution mass spectrometry method using capillary gas chromatography negative ion chemical ionization for the determination of cortisol in human plasma. Cortisol was derivatized with pentafluoropropionic anhydride at room temperature and measured by selected ion monitoring of the molecular ion. This method utilized d4-cortisol as an internal standard and required only a simple correction for the isotope contribution. A sensitivity as low as 0.02 pmol (s/n > 10) can be achieved easily. Therefore a plasma sample as small as 0.1 ml can be used for the accurate measurement of cortisol in humans.