Melatonin is an indole hormone secreted by the pineal gland during the hours of darkness in a normally entrained individual. There is a clear circadian rhythm in its production with low levels during the day and a peak in the early hours of the morning. The timing of sample collection is crucial and single time point measurements are of little use. Measurement of melatonin or its major metabolite, 6-sulphatoxymelatonin, is normally carried out to determine the timing of an individual's internal body clock and whether it is synchronized to the 24 h day. Misalignment of the clock or disruption of the rhythm can lead to difficulties in sleeping and health problems such as are associated with jet-lag or shift work. Both melatonin and 6-sulphatoxymelatonin can be measured by RIA or ELISA. Details of sample collection and preparation and the assay procedures are described.