Mushrooms are a food with high nutritional value. Antrodia camphorata (AC), is a medicinal mushroom being widely used as food dietary supplement for cancer prevention. The sesquiterpene lactone, antrocin reported as a novel dual Akt and mTOR inhibitor in metastatic breast cancer cells and, is the most potent among more than one hundred secondary metabolites isolated from AC. For the first time, this study developed and validated a simple high-performance liquid chromatography coupled with diode-array detector (HPLC-DAD) method for determination of antrocin in AC extracts. Separation of antrocin was achieved within 8 min on a J，sphere ODS-M80 C18 column using the gradient mobile phase acetonitrile and water containing 0.1% formic acid with a flow rate of 1.0 mL/min and DAD detection at 205 nm. The method produced linear response in the concentration range of 100-1600 昭/mL with a detection limit of 64.3^g/mL and a quantification limit of 194.8 |ig/mL. The method was validated in terms of intra- and inter-day precision (within 6.3% and 8.9%, respectively). At the fortified levels of 200, 400, 600 and 800 |ig/mL, the mean recoveries of antrocin ranged from 98.4% to 101.2%. The developed method successfully was applied for the determination of antrocin in AC preparations.