Abstract: We isolated and purified the component of 9-oxo-10,11-dehydroagerophorone (Euptox A), a hepatotoxin and an insecticide constituent, from Eupatorium adenophorum leaves by silica gel column chromatography and reverse-phase high performance liquid chromatography (HPLC). We developed an HPLC method for determining Euptox A from E. adenophorum, which was confirmed by HPLC-DAD-MS. The chromatographic separation was achieved on a Cosmosil Rp C₁₈ column with methanol:water (60:40, v/v) as the mobile phase, flow rate 0.8 mL·min^–1 and wavelength 255 nm. Data were corroborated by HPLC-MS with electro-spray ionization of positive ion mode. The limit of detection for Euptox A was 0.4 μg·g^–1. The recoveries were 97.3%–103.7% and relative standard deviations 2.4%–3.5%. Euptox A was mainly distributed in leaves (1.34–8.41 mg·g^–1) and only a small amount was detected in flowers, stems, or roots (< 0.06 mg·g^–1). Euptox A mainly accumulated in plant leaves in the vegetative period (from May to October), with less accumulation in the reproductive period (from November to the next April). The established HPLC method is simple, quick and could be used for component determination in E. adenophorum products for quality control and safety evaluation.