Late-night salivary cortisol (LNSC) is now considered the best approach to screen patients suspected of having endogenous hypercortisolism (Cushing's syndrome). As the use of LNSC increases, new preanalytic and analytic issues have arisen. The routine immunoassay for salivary cortisol seems to have better diagnostic performance than liquid chromatograph/tandem mass spectrometry, although measurement of normal salivary cortisone concentrations with the latter technique is very useful in identifying samples contaminated with topical hydrocortisone. LNSC is very useful in screening for Cushing's syndrome in women with increased corticosteroid-binding globulin resulting from estrogen therapy or pregnancy. Two LNSCs from each patient is recommended for routine screening, although one adequate saliva sample seems to perform well. The overnight dexamethasone suppression test remains superior to LNSC in the evaluation of potential subclinical hypercortisolism in patients with adrenal incidentalomas. Periodic assessment of LNSC is extremely useful in monitoring patients for recurrence of Cushing's disease after pituitary surgery. With the large increase in the number LNSCs being ordered around the world, it is likely that more preanalytic and analytic issues will arise, which laboratorians and clinical chemists will need to resolve.