A simple and highly sensitive method was developed for the determination of organic acids exuded from plant roots by high performance liquid chromatography with ultraviolet (UV) detection. The root exudate was passed through cation-exchange column and subsequently through anion-exchange resin column. The eluate was then concentrated in a rotary evaporator. The residue was dissolved in dilute HClO 4 solution at pH 2.1. The separation was performed on a Bio-Rad Aminex HPX-87H sulfonic column at 50 °C with an eluent containing 5 mmol/L H 2 SO 4 at a flow rate of 0.5 mL/min, and the organic acids were detected at 210 nm by a UV detector. Average recoveries for the root exudates were in the range of 82.0%–96.2% and the detection limits were 1–120 μg/L, and the relative standard deviations (RSDs) were 0.67%–3.31% for the seven organic acids. The intraday precisions were 1.2%–4.7%, and interday precisions were 3.3%–10.6%. These results demonstrated that the proposed method is simple, sensitive, and reliable for the determination of organic acids in plant root exudates, despite the presence of the particularly complex matrix.