There is considerable variation in the methods used to diagnose and investigate adrenal insufficiency in clinical practice. These include a range of adrenocorticotropin (ACTH) stimulation and other dynamic testing protocols, serum cortisol cut-off values for diagnosis and tests used for differential diagnosis. With the introduction of modern cortisol and ACTH assays, the interpretation of tests used for diagnosis and differential diagnosis has become more complex and requires local validation. This review examines the basis of normal hypothalamic-pituitary-adrenal axis function and adrenal insufficiency states based upon an evidence base accumulated over the past four decades. The role of the laboratory in the differential diagnosis and interpretation based upon assay methodology is discussed. The accurate identification of patients who may benefit from corticosteroid replacement in special settings such as critical illness is challenging and will be explored.