Because of the variability in the clinical expression of Cushing's syndrome, different biochemical tests and imaging techniques must be used for effective diagnosis and treatment. The clinical history is important to assess the general impact of hypercortisolism on organs and systems, as well as to guide suspicion toward more aggressive entities such as overt ectopic ACTH syndrome or to detect an iatrogenic etiology of Cushing's syndrome. The screening phase relies on tests that are sensitive but not specific, such as urinary free cortisol, nocturnal salivary cortisol and 1 mg dexamethasone suppression, which still require established assessment criteria. Confirmation can be obtained by repeating urinary cortisol, 2 mg/day dexamethasone suppression, both alone and combined with corticotropin releasing hormone (CRH), to exclude pseudoCushing states. ACTH dependency can be easily explored by ACTH measurements, but the differential diagnosis between pituitary and ectopic Cushing's disease is difficult and may require invasive procedures such as inferior petrosal sinus sampling. Sophisticated imaging and isotopic techniques play a significant role in locating the source of ACTH in ectopic syndromes but are not always effective. In general, biochemical and imaging tests should be combined in order to assess different mechanisms and perspectives of the syndrome. Rigorous methodology is essential to obtain accurate results, allowing a correct diagnosis and improving therapeutic performance in this devastating disease.