The Euphorbiaceae family comprises of about 300 genera and 5000 species primarily distributed in America and tropical Africa. The Euphorbia genus is represented by 105 species and locally named as "Sutlegen" and "Xasil" in Turkey. The present study aimed to determine the chemical constituents of E. aleppica, E. eriophora, E. macroclada, E. grisophylla, E. seguieriana subsp. seguieriana, E. craspedia, E. denticulata, E. falcata, and E. fistulosa, and classify them by utilizing the chemometric techniques of principal component analysis (PCA) and hierarchical cluster analysis (HCA). Linoleic acid, 17-tetratriacontane, palmitic acid, and hexatriacontane were the major fatty acids from the gas chromatography-mass spectrometry (GC/MS) analyses. Characterization of 268 constituents of the studied species was achieved by liquid chromatography-ion trap-time-of-flight-mass spectrometry (LC-IT-TOF-MS). Furthermore, a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous quantitative determination of 11 compounds (quinic acid, protocatechuic acid, rutin, hesperidin, eugenol, p-coumaric acid, piceatannol, scopoletin, dl-kavain, chrysophanic acid, and resiniferatoxin) in these species. The developed method was validated for the linearity, limit of detection, limit of quantification, repeatability, and recovery.