Melatonin functions as a free radical scavenger and controls the regulation of the sleep–wake cycle in mammals, while serotonin is the main intermediate in melatonin biosynthesis. In this paper, melatonin and serotonin have been detected and quantified for the first time in eight different sweet cherry cultivars using high performance liquid chromatography with mass spectrometry detection. The limits of detection of the proposed analytical method were 4.3 ng/mL for melatonin and 3.0 ng/mL for serotonin. An inverse relation between the contents of melatonin and serotonin was observed in the studied sweet cherry cultivars. The highest melatonin amounts were found in ‘Burlat’ sweet cherries (22.4 ± 1.3 ng/100 g of fresh fruit), while the highest serotonin contents were found in the cultivar ‘Ambrunés’ (37.6 ± 1.4 ng/100 g of fresh fruit). The results presented in this research allow us to know the amount of melatonin and serotonin bring to the diet.