Cortisol is the primary glucocorticoid in humans, affecting glucose control, inflammation, and respond to stress. Cortisol stimulates gluconeogenesis in the liver and reduces insulin secretion while increasing glucagon release by the pancreas. This increases blood glucose levels. Cortisol is also involved in inhibiting inflammatory responses, and maintaining blood pressure by potentiating effects on norepinephrine. Most of circulating cortisol is bound to protein, primarily transcortin (corticosteroid binding globulin, CBG) and albumin. The free hormone hypothesis suggests that the unbound, or free, cortisol is the active fraction, and that this fraction is the most important clinically. Total serum cortisol may be an adequate measure of cortisol activity except when the levels of the binding proteins are abnormal such as in liver disease or acute illness. A study published in the New England Journal of Medicine demonstrates the utility of measurements of free cortisol in critically ill patients. Patients with critical illness increase cortisol secretion, however this is best observed when free cortisol levels are measured. In the study 40% of patients with hypoproteinemia had low levels of total cortisol even though their adrenal function was adequate as demonstrated by robust response to ACTH. Similar results were obtained when salivary cortisol was used as a marker for adrenal sufficiency during illness. A number of tests to determine free cortisol have been devised. The free fraction depends on the concentrations of the binding proteins and cortisol, and, thus, may be calculated based on these factors. However, free cortisol is best is best measured by equilibrium dialysis. Structure-function observations favor a direct measure of free cortisol. There are polymorphic forms of transcortin that affect cortisol binding, and glycosylation affects cortisol binding to transcortin. The CORTISOL, FREE EQUILIBRIUM DIALYSIS AND LC/MS-MS assay provides a specific direct test.