A rapid, isocratic high performance liquid chromatographic(HPLC) method with fluorescence detection was used for the determination of total homocysteine (Hcy) levels in human plasma. Prior to reversed-phase HPLC analysis, the plasma thiols were derivatized with ABDF(7-fluoroben30-2-oxa-1,3-diazole-4-sulfonamide), a thiol specific fluorogenic probe. A mobile phase consisted of 0.1 mol/L potassium dihydrogen phosphate (pH 2.1, containing 10% of acetonitrile) ensured base line separation of plasma Hcy. The mean recovery of this method was 97.75%. The intraday and interday coefficient of variations were 4.49% and 9.79%, respectively. The method is simple, sensitive, reproducible and very suitable for routine determination of plasma Hcy in human.