A patient was diagnosed with Cushing's syndrome during her first pregnancy. Bilateral simultaneous inferior petrosal sinus corticotropin sampling with corticotropin-releasing hormone stimulation was performed before transphenoidal pituitary adenomectomy, with successful localization of the pituitary adenoma. Her Cushing's syndrome was controlled postoperatively with resolution of hypertension. This case report demonstrates that the procedure of bilateral simultaneous inferior petrosal venous corticotropin sampling can be safely performed during pregnancy.