OBJECTIVE: To establish a HPLC assay for the determination of idebenone in human plasma. METHOD: The method used reversed phase C18 column and methanol-water (70:30) as mobile phase, with UV detection at 280 nm. To the plasma samples, 6 mol·L-1 HCl were added and then extracted by diethyl ether. After evaporating diethyl ether on a water bath under nitrogen, the residues were dissolved by methanol and injected into the HPLC system. The plasma samples of two healthy volunteers were determined. RESULTS: The calibration curve was linear within the range of 20~1600 ng·ml-1 and the limit of quantitation was 5 ng·ml-1. The average recovery of idebenone in samples of 40, 200, 800 ng·ml-1 was 73.86%, 83.72% and 76.17%, respectively. The RSD for the inter- and intra-day was less than 5.38%. CONCLUSION: The method was a simple, sensitive and accurate method. It is suitable for the monitoring of blood drug concentration and for the purpose of bioavailability study.