Infusions or dried leaves from Morus alba (L., Moraceae) have long been used in folk medicine for the prevention or treatment of several diseases. Despite the great interest in determining the role of phytonutrients as potential therapeutic agents, and the rising demand of natural sources with nutraceutic benefits, the antioxidant content of many foodstuffs is unknown, making accurate estimation for human dietary consumption and the correlation to human diseases difficult. The high content in polyphenols mainly accounts for in vitro and in vivo antioxidant activity of extracts obtained from this plant, and it is responsible for their preventive and therapeutic roles in a number of human diseases. In this study, we achieved full characterization and quantitation of flavonoids and other phenolic components extracted from lyophilized mulberry leaves, by maceration with ethanol. To tackle such a task, an RPLC-diode array detector-MS system was optimized, employing a partially porous octadecylsilica column as stationary phase. Identification of mulberry leaves constituents was carried out on the basis of the complementary information obtained from their migration times, diode array spectra, MS ions, and MS/MS fragments. The employment of the hybrid (IT-TOF) mass spectrometer further allowed the structural assignment of a series of structural isomers. To the best of our knowledge, 11 out of 22 identified compounds are being reported in mulberry leaves for the first time, and the relative amounts determined, in the two cultivars analyzed.