Objective: Cortisol is an important hormone/steroid in the regulation of intermediate metabolism and stress. It exists in free (unbound) and protein-bound forms in serum but only in a free form in saliva. The free form is the biologically active one. There is an advanced biofeedback system regulating the cortisol secretion/concentration by the hypothalamic-pituitary-adrenal axis. Methods: There are many different methods to collect saliva, either directly from the mouth as an expectorate or indirectly by using swabs, capillary tubes or paper foams. The swab may be cotton-based, synthetic, with or without a plastic sheath. There are different methods for analysis – immunological, radioisotopic, mass fragmentographic and chromatographic. Results: The diurnal variation in salivary cortisol concentrations in healthy school-aged boys and girls are described and show dependence to different degrees of age and sex. Conclusion: Sampling for salivary cortisol determination is a sensitive, specific and pain-free procedure and it is therefore very useful for following healthy and sick children before, during and after pharmacological interventions. Different conditions as time for sampling, interaction with other endogenous and exogenous steroids, and physical and psychological activity interact with the salivary cortisol concentration.