The diagnostic evaluation of Cushing's syndrome proceeds in two stages: first the definitive diagnosis of pathological hypercortisolism (Cushing's syndrome), and second the differential diagnosis of pituitary, adrenal, and ectopic causes. Proper initial diagnosis of Cushing's syndrome depends on correlation of clinical data with results of 24-hour urine free cortisol and responses to low-dose dexamethasone suppression. Although responses to corticotropin-releasing hormone (CRH) administration differ between patients with Cushing's syndrome and those with pseudo-Cushing's states such as stress, depression, and alcoholism, this test is not, at present, useful in the initial diagnosis of Cushing's syndrome. When the diagnosis of Cushing's syndrome has been secured, measurement of ACTH or β-LPH provides a reliable distinction between ACTH-dependent and adrenal-based etiologies. New criteria for the standard 48-hour high-dose dexamethasone test identify approximately 80% of patients with Cushing's disease. The CRH stimulation test may provide additional diagnostic accuracy. Inferior petrosal sinus sampling is a highly accurate test for ACTH-dependent Cushing's syndrome when other test results are inconclusive.