A method was established for the determination of deoxyspergualin using reversed-phase ion-pair chromatography (RP-IPC). Several parameters, such as the chromatographic column, the types and concentration of ion-pair reagents, the concentration of the buffer and the pH value of the mobile phase were evaluated. The optimal separation conditions were as follows: C18 column (250 mm x 4.6 mm, 5 microm); mobile phase, 5 mmol/L di-potassium hydrogen phosphate (containing 5 mmol/L 1-pentanesulfonic acid sodium, pH 3.6 +/- 0.3)-acetonitrile (90: 10, v/v); flow rate, 1.0 mL/min; detection wavelength, 210 nm; column temperature, 30 degrees C; injection volume, 20 microL. Under the optimaized experimental conditions, good linear relationships was obtained and the limit of detection for deoxyspergualin was 0.5 mg/L.