Background: In patients with adrenal incidentalomas (AIs), there is uncertainty on how to rule out hypercortisolism. The occurrence of postsurgical (unilateral adrenalectomy) hypocortisolism (PSH) has been proposed as a proof of the presence of presurgical hypercortisolism in AI patients. The aim of this study was to define the thresholds of cortisol level after the 1 mg overnight dexamethasone suppression test (F-1mgDST), urinary free cortisol (UFC), midnight serum cortisol (MSC), and adrenocorticotropin (ACTH) to predict the absence of PSH in AI patients undergoing surgery. Methods: In 60 patients who underwent AI excision, cortisol secretion was assessed by a low-dose corticotropin stimulation test or insulin tolerance test when needed. We searched for the lowest presurgical value of F-1mgDST, UFC, and MSC and the highest value for ACTH in AI patients with PSH as indexes of normal cortisol secretion. Results: The lowest values of F-1mgDST, UFC, and MSC and the highest value for ACTH in PSH patients were 1.2 µg/dL (33 nmol/L), 10.4 µg/24 hours (29 nmol/24 hours), 1.2 µg/dL (33 nmol/L), and 26.9 pg/mL (6 pmol/L), respectively, but only F-1mgDST <1.2 µg/dL (33 nmol/L) was able to predict the absence of PSH. Among AI patients with F-1mgDST <1.2 µg/dL (33 nmol/L) no subjects had diabetes mellitus and/or metabolic syndrome, and these subjects tended to have a better metabolic profile than those with F-1mgDST ≥1.2 µg/dL (33 nmol/L) Conclusion: In AI patients a F-1mgDST <1.2 µg/dL (33 nmol/L) rules out PSH and could be used to exclude hypercortisolism in AI patients.