Hyphaene thebaica is an ancient palm whose fruits are an integral part of the human diet. An approach based on high-performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC-ESI-MS) in the negative ion mode, evidencing phenolic molecules as the main constituents of the plant has been developed. These phenolic molecules, widely distributed in plants and considered highly beneficial for their antioxidant properties, are very difficult to separate and characterize. Their presence has been rationalized on the basis of their mass spectrometry profiles. A Thermo Surveyor BPLC system and a C18 reversed phase column are used, and the mobile phase with a mixture of acetonitrile and 0.03% formic acid (v/v) in water in the gradient elution mode at flow rate 0.4 mL/min, detected at 360 nm. Phenolic molecules are screened based on accurate mass of pseudomolecular EM-HT ions and tandem mass spectrometry (MS/MS) data. The data obtained in our research show that Hyphaene thebaica fruits are a rich source of phenolic acids and flavonoids with evident of strong antioxidant.