Dronedarone, a noniodinated benzofuran derivative of amiodarone, is believed to have a better side effect profile, and is currently undergoing phase III clinical trials. A novel method was developed for the determination of dronedarone and its principal metabolite debutyldronedarone in both plasma and myocardial tissue by high-performance liquid chromatography (HPLC) coupled with UV-detection. The assay was also validated for determination of amiodarone and desethylamiodarone. Samples were obtained from healthy humans (plasma) and goats (plasma and myocardium). Sample preparation included deproteinization with acetonitrile and extraction with a mixture of heptane and dichloromethane (50/50, v/v). Chromatographic separation was performed on a Pathfinder PS polymeric C18 column (50 mm x 4.6 mm, 2.5 microm) with a mobile phase of acetonitrile, isopropanol, water and ammonia (80/10/10/0.025, v/v/v/v) at a flow-rate of 1 ml/min. Calibration curves of all analytes were linear in the range of 0.01-5 microg/ml for plasma samples, with a lower limit of quantification (LLOQ) of 0.04 microg/ml. For myocardial tissue samples, linear curves of all analytes were observed in the range of 0.02-500 microg/g, with a LLOQ of 0.08 microg/g. Within- and between-day precision was <18%, and within- and between-day accuracy ranged from 97.5 to 109.7%, with a recovery of 67.6-79.9%. The present method enables sensitive and specific detection of dronedarone, amiodarone and principal metabolites in plasma as well as myocardial tissue.