Serum cortisol levels do not provide high-end diagnostic accuracy or sensitivity when used as an initial diagnostic test. Late-night salivary cortisol samples employing an approved collection device, 24-hour urine-free cortisol, or a 1-mg overnight dexamethasone suppression test should be used as an initial test. These tests have a high diagnostic accuracy for Cushing syndrome. Multiple screening tests may need to be performed based on the variability of hypercortisolism in Cushing syndrome. Two measurements of abnormal cortisol levels with these tests are recommended for an initial diagnosis; further workup should be referred to an endocrinologist to make the final diagnosis. Each of these tests has different limitations and should be chosen based on the lifestyle and medical history of the patient. Patients with erratic sleep schedules or shift workers would not obtain accurate results from a late-night salivary cortisol test. Women taking oral estrogen, those taking antiepileptic drugs (e.g., phenytoin and phenobarbitone), and pregnant women could have falsely elevated cortisol levels as CYP3A4 metabolizes dexamethasone with the dexamethasone suppression test. Urine-free cortisol tests require rigorous collection management and should not be used on patients experiencing renal failure, or those suspected to have mild Cushing syndrome. Exogenous glucocorticoid use must be excluded before performing these biochemical tests.