Inferior petrosal sinus sampling (IPSS) helps differentiate the source of ACTH-dependent hypercortisolism in patients with inconclusive biochemical testing and imaging, and is considered the gold standard for distinguishing Cushing disease (CD) from ectopic ACTH syndrome (EAC). We present a comprehensive approach to interpreting IPSS results by examining several real cases.We performed a comprehensive review of the IPSS literature using PubMed since it was first described in 1977.IPSS cannot be used to confirm the diagnosis of ACTH-dependent Cushing syndrome (CS). It is essential to establish ACTH-dependent hypercortisolism prior to the procedure. IPSS must be performed by an experienced interventional or neuroradiologist as successful sinus cannulation relies on operator experience. In patients with suspected cyclical CS it is important to demonstrate the presence of hypercortisolism before IPSS. Concurrent measurement of IPS prolactin (PRL) levels is useful to confirm adequate IPS venous efflux. This is essential in patients who lack an IPS-to-peripheral (IPS:P) ACTH gradient, suggesting an ectopic source. The prolactin-adjusted IPS:P ACTH ratio can improve differentiation between CD and EAS when there is a lack of proper IPS venous efflux. In patients who have unilateral successful IPS cannulation, a contralateral source cannot be excluded. The value of the intersinus ACTH ratio to predict tumor lateralization may be improved using a prolactin-adjusted ACTH ratio, but this requires further evaluation.A stepwise approach in performing and interpreting IPSS will provide clinicians with the best information from this important but delicate procedure.