In this work, the phytochemical profile of the ethanolic extract of Euphorbia peplus L. collected in Central Italy, was reported. This specimen had never been studied before and the analysis was accomplished by means of Column Chromatography for the separation procedure and by means of NMR Spectroscopy and Mass Spectrometry for the identification step. In particular, fourteen compounds were evidenced belonging to five different classes of natural compounds i.e. triterpenoids (pentacyclic and saponin), peculiar diterpenoids (jatrophanes and pepluanes), flavonoids (flavonols), caffeoyl-quinic acids and rare disaccharides. In addition to this, a semi-quantitative analysis on the diterpenoid fraction, by means of NMR Spectroscopy, was also performed in order to provide the real quantities of these compounds in the same fraction and in the total extract. Due to the pronounced chemo variability observed in Euphorbia spp., the availability of a reliable and quick analytical technique, such as that reported in the present study, could be a useful tool in the standardization of plant materials to be used in pharmacological studies or for ethnomedicinal purposes. The technical details for both the general phytochemical analysis and the specific quantitative one, were inserted in this paper. Moreover, the chemotaxonomic and ethnopharmacological relevance of these compounds was also discussed.