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Inferior petrosal sinus sampling (IPSS) is a specialized medical procedure primarily utilized for the assessment of Cushing's disease, a condition characterized by excess cortisol production often caused by a pituitary adenoma. This technique involves the selective catheterization of the inferior petrosal sinuses, which are venous channels located at the base of the skull and are important for draining blood from the pituitary gland. The procedure is minimally invasive and typically performed under local anesthesia with the assistance of fluoroscopy or CT guidance. During IPSS, catheters are inserted into the inferior petrosal sinuses via the internal jugular vein to measure hormone levels, particularly adrenocorticotropic hormone (ACTH), in the blood that drains from the pituitary gland. By comparing the ACTH concentrations in the blood from these sinuses to those measured in the peripheral blood, clinicians can determine whether the source of increased hormone production is indeed a pituitary tumor (Cushing's disease) or if it arises from other origins, such as ectopic ACTH syndrome or adrenal adenomas. A distinctly elevated gradient of ACTH from the inferior petrosal sinuses compared to the peripheral blood provides strong evidence of a pituitary source of excess ACTH and supports the diagnosis of Cushing's disease. This diagnostic modality is critical because it influences subsequent treatment decisions, which may involve surgical resection of the pituitary tumor, medical therapies, or radiation. Moreover, IPSS is particularly valuable in complicated cases where non-invasive imaging techniques, such as MRI, may render inconclusive results. Besides the diagnostic advantages, this procedure is associated with minimal complications, and the risks are generally low, including potential bleeding, infection, or vascular complications related to catheter placement. However, the experience of the medical team and the facility performing the procedure is pivotal to its success and safety. Following the procedure, patients are monitored for a short period to ensure stability before discharge. Ultimately, inferior petrosal sinus sampling stands as an essential tool in the endocrinologist's arsenal, facilitating accurate diagnosis and management of hypercortisolism while helping discern the cause of hormonal dysregulation. Its significance lies not only in enhanced diagnostic accuracy but also in being a pivotal step towards targeted and effective treatment strategies for individuals affected by Cushing's disease and related disorders.