For examination of glucocorticoid metabolism and identification of hyper and hypocortisolism, various measurements and diagnostic tools are available. After a brief overview of the physiology of glucocorticoid secretion and glucocorticoid actions, the currently used measurements for blood, saliva, and urine samples and the corresponding physiological and metabolic implications are critically reviewed. A special emphasis is placed on the potential of 24-h urine analyses to assess not only glucocorticoid secretion, but also functional glucocorticoid activity.