Globularia alypum L. (Globulariaceae) is a plant growing in the Mediterranean basin and is known and used in the folk medicine for its several pharmacological properties against typhoid, fever, gout, diabetes, and rheumatism. The methanol extract of Globularia alypum has been characterized for its phytochemical composition using the liquid chromatography hyphenated with an ESI-QTOF-MS. Thus, in this work the used technique leads to the tentative characterization of a total of 60 phytochemical compounds. The major compounds identified belong to the iridoids and phenolic acids derivatives. More than 20 iridoids and secoiridoids were identified, including several compounds not previously documented in Globularia alypum, such as gentiopicroside, acetylbarlerin isomers, serratoside A, specioside, shanziside, and decumbeside D isomers. Besides, the presence of 10 flavonoids, together with other nine polar compounds, has been confirmed. The methanol extract of Globularia alypum could thus be considered as a promising source of bioactive ingredients promoting the further use of this plant in the folk medicine.