The assessment of cortisol in saliva has proven a valid and reliable reflection of the respective unbound hormone in blood. To date, assessment of cortisol in saliva is a widely accepted and frequently employed method in psychoneuroendocrinology. Due to several advantages over blood cortisol analyses (e.g., stress-free sampling, laboratory independence, lower costs) saliva cortisol assessment can be the method of choice in basic research and clinical environments. The determination of cortisol in saliva can facilitate stress studies including newborns and infants and replace blood sampling for diagnostic endocrine tests like the dexamethasone suppression test. The present paper provides an up-to-date overview of recent methodological developments, novel applications as well as a discussion of possible future applications of salivary cortisol determination.