Establishing the cause of Cushing's syndrome (CS) is one of the most challenging processes in clinical endocrinology. Biochemical testing, including measurement of plasma adrenocorticotropin (ACTH), high-dose dexamethasone suppression, and corticotropin-releasing hormone stimulation testing, is integral to the differential diagnosis. No existing test has sufficient diagnostic accuracy when used alone, however. The adjunctive use of focused imaging, including CT, MRI, and nuclear imaging modalities, often can provide a diagnosis. In patients with ACTH-dependent CS, bilateral inferior petrosal sinus sampling can facilitate a diagnosis in those with discrepant clinical features, biochemistry, or imaging. This article focuses on current biochemical and radiologic strategies for the differential diagnosis of CS.