BACKGROUND Various species of the Euphorbia genus contain diterpene ingenol and ingenol mebutate (ingenol-3-angelate), a substance found in the sap of the plant Euphorbia peplus and an inducer of cell death. A gel formulation of the drug has been approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the topical treatment of actinic keratosis. OBJECTIVE To develop a rapid and reliable method for quantification of ingenol in various plant extracts. METHODS Methanolic extracts of 38 species of the Euphorbia genus were analysed via ultra-high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) after methanolysis and solid-phase extraction (SPE) purification. The 18 O-labelled ingenol analogue was prepared and used as an internal standard for ingenol content determination and method validation. RESULTS The highest ingenol concentration (547 mg/kg of dry weight) was found in the lower leafless stems of E. myrsinites. The screening confirms a substantial amount of ingenol in species studied previously and furthermore, reveals some new promising candidates. CONCLUSIONS The newly established UHPLC-MS/MS method shows to be an appropriate tool for screening of the Euphorbia genus for ingenol content and allows selection of species suitable for raw material production and/or in vitro culture initiation. Copyright © 2017 John Wiley & Sons, Ltd.