Spontaneous hyperadrenocorticism is a common and well-recognised endocrine disorder occurring in dogs. The biochemical diagnosis of hyperadrenocorticism rests on the documentation of excessive glucocorticoid levels or metabolites in urine or blood. The diagnosis of canine hyperadrenocorticism is historically developed from human methods and based on non-dynamic and dynamic tests and visualisation. In some cases the different ranges of endocrine parameters or metabolites necessitate the modification of human protocols. Elevation of the activity of alkaline phosphatase (AP) and its heat-resistant isoenzyme (SIAP) induced by endogenous or exogenous glucocorticoid excess raise the suspicion of hyperadrenocorticism. Cortisol values in morning urine are related to creatinine concentrations to correct for differences in urine concentration. Theoretically, the administration of dexamethasone (DX) at a relatively low dose (0.01 mg/kg of body weight) can inhibit the pituitary secretion of ACTH and, in turn, decrease endogenous cortisol secretion for as long as 24 to 48 h. Therefore, DX administration to dogs with a functioning adrenocortical tumour would not affect the plasma cortisol concentration at any time following its administration. The high-dose dexamethasone suppression test (HDDS) is based on the observation that the function of adrenocortical tumours is independent of pituitary ACTH and they completely suppress ACTH secretion; therefore, regardless of its dose, dexamethasone is never able to suppress cortisol secretion. HDDS can be combined with the measurement of urinary cortisol/creatinine (c/c) ratio from morning urine samples on three consecutive days. In case of non-suppressible urinary c/c ratio one has to speculate on differentiating adrenal tumour (AT) from non-suppressible pituitary-dependent hyperadrenocorticism (PDH) due to a pituitary tumour arising from the intermediate lobe. Radiocholesterol scintigraphy is a less frequently used technique in the diagnosis of canine Cushing's syndrome (CCS); however, it has the same advantages in the localisation and characterisation of adrenocortical diseases as in humans.