Bilateral inferior petrosal sinus sampling is the gold standard diagnostic assay in identifying the pituitary source of adrenocorticotropic hormone secretion in Cushing syndrome. The noninvasive assays used in the workup of Cushing syndrome are often misleading or ambiguous, yielding sensitivities of up to only 80%. Inferior petrosal sinus sampling, however, is highly accurate and safe when performed in experienced centers. We review here the historical and technical details of the procedure, with emphasis on the relevant anatomy and a discussion of possible rare complications.