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Melatonin supplements are used widely in Europe and the United States (US) to alleviate jet-lag and other sleep-delay disorders. Recently, the European Commission authorized two health claims for food containing melatonin, with a minimum dose per quantified portion. This study aimed to determine the dose of melatonin in food supplements marketed in Europe (Spain) and the US by validating a liquid chromatography method with diode array detection (LC-DAD). Additionally, contaminants present in melatonin supplements were analyzed using ultra-high-performance liquid chromatography coupled to an orbitrap mass spectrometer. We tentatively identified 8 tryptophan-related contaminants in melatonin supplements, suggesting tighter impurity control in melatonin supplements is necessary. The LC-DAD validated method achieved reliable results regarding recovery, precision, repeatability, limit of detection, limit of quantitation and linearity. Label melatonin dose varied from 1–1.95 mg/unit and 0.3–5 mg/unit for supplements marketed in Europe (Spain) and the US, respectively. Four out of seventeen supplements showed significant deviations from melatonin content declared on the label (from −60% to −20%). Regarding health claims, three out of the ten supplements purchased in Spain should not include the claim “reduction of time taken to fall asleep”, as their melatonin content is lower than 1 mg. [ABSTRACT FROM AUTHOR]