Venous sampling is the gold standard for localizing abnormal hormone secretion in several endocrine disorders. The most common indication for venous sampling is in the workup of primary aldosteronism, adrenocorticotropic hormone-dependent Cushing’s syndrome, and hyperparathyroidism. In experienced hands, venous sampling is safe and accurate. This review discusses the role of venous sampling in the workup of endocrine disease, describing the underlying anatomy and pathophysiology, as an understanding of these concepts is essential for technical and clinical success.