Abstract: The strong antioxidant activity of melatonin is well known and it is important to investigate its presence and levels in different foodstuffs, for the purpose of evaluating their nutraceutical properties. As a contribution towards this goal, an original analytical method has been developed for the simultaneous determination of melatonin and other indolic and phenolic antioxidants (including trans- and cis-resveratrol, ferulic acid, tryptophan, serotonin and 5-hydroxyindoleacetic acid) in grape-related foodstuffs and beverages: namely grape, grape juice, must, wine and grappa (Italian pomace brandy). These foodstuffs represent an important part of the diet, both traditionally and in recent times, especially in Mediterranean countries and could be (at least in part) responsible for the beneficial effects involved in the ‘French paradox’. The analytical method is based on high-performance liquid chromatography coupled to fluorescence detection, exploiting the native fluorescence of the analytes. A C8 column was used as the stationary phase, while the mobile phase was composed of acidic phosphate buffer and acetonitrile; fluorescence intensity was monitored at λ = 386 nm while exciting at λ = 298 nm. The sample pretreatment was carried out by a fast and reliable microextraction by packed sorbent (MEPS) procedure. After validation, the method was applied to the analysis of melatonin and other antioxidants in food and beverages derived from grape, with very good results being obtained. Thus, this methodology may represent a promising tool for the evaluation of the antioxidant properties of nutraceuticals and functional foods.