ABSTRACT Nowadays, several groups are devoted to the development of reliable analytical methodologies such as microchip electrophoresis (ME), capillary electrophoresis (CE), liquid chromatography (LC), coupled to different detection strategies, and, particularly, the application of nanotechnologies and nanomaterials in the development of sensors for the determination of melatonin (MEL), its isomers, and precursors in different plant tissues and food samples. These approaches provide useful tools and knowledge to demonstrate the great potential of this natural substance for agricultural applications, improving crop production and quality of functional foods. The analysis of MEL in plants and foods represents a highly challenging task due to its low concentration range, the difculty in the selection of the extraction solvents because of its amphipathic nature, and the fact that it reacts quickly with other matrix components. Chemical complexity of plant extract can also interfere with MEL determinations, giving false positive results if methods from vertebrate MEL research are directly adopted. Indeed, the additional challenges related to the vast variety of matrices as well as the extremely wide concentration range (ppm-ppq levels) should be considered. Therefore, the main objective of this chapter is to take a closer look at the recent trends in analytical tools employed to contribute to the elucidation of the biological role of MEL in plants, and its implications in human health.