The overview of cortisol physiology, action and pathology is achieved in relation to the hypothalamic-pituitary-adrenal axis alteration by laboratory investigation. The measurements of cortisol and related compound levels in blood, urine and saliva used to study the physiological and pathological cortisol involvement, are critically reviewed. The immunoassay and chromatographic methods for cortisol measurement in the various biological fluids are examined in relation to their analytical performances, reference ranges and diagnostic specificity and sensitivity. Moreover, blood, urine and saliva cortisol level measurements are described taking into account the diagnostic implications. The deduction is that each method requires the definition of its own reference range and its related diagnostic cut-off levels. Thus, this review, stressing the analysis procedures, could help to understand and compare the results of the different assays.