Melatonin, a powerful antioxidant, offers potential human benefits in the fields of medicine, nutrition, and food science. While best understood in a mammalian system, melatonin has been identified in plants and dietary melatonin has been shown to increase circulating levels in the blood. Thus, there exists a great interest in extracting and detecting melatonin present in edible plant matrices. Extraction techniques such as liquid/liquid, solid phase, and solid/liquid extraction were investigated and compared to determine the best approach for isolating melatonin from fruit. Enzyme-linked immunoassay (ELISA), fluorescence, and mass spectrometry were investigated for their use as detection methods for melatonin originating in fruit systems. Additionally, the stability of melatonin in a pH 3.5 buffered model system was studied to gain preliminary information regarding melatonin heat and light stability. It was determined that melatonin is both heat stable and light stable for up to one hour (85C and 17 par, investigated separately). Solid/liquid extraction using ethyl acetate as a solvent was determined to be the best extraction procedure while high performance liquid chromatography-mass spectrometry with the use of a deuterated internal standard was the preferred detection method. A significant amount of work remains in the area of quantification of melatonin from fruit systems.