Cortisol is one of the most important glucocorticoids and plays important roles in regulating human metabolism. Midnight salivary cortisol has been shown to correlate well with free cortisol concentration in serum and is one of the first tests recommended for the diagnosis of Cushing's syndrome. The procedure described here involves centrifugation of the saliva samples to remove solids and mucus strands before they are diluted with buffer and mixed with deuterated internal standard D4-cortisol. The samples are then subjected to reverse phase separation on a C18 column and analyzed by a tandem mass spectrometry method (LC-MS/MS). Quantification is achieved by comparing the responses of a given sample to the responses of the calibrators of known concentrations. The calibrators are prepared and analyzed along with the patient samples. Analytical specificity is ensured by using multiple reaction monitoring with fragment ions that are unique to cortisol and deuterated internal standard.