A screening method for asparagine synthetase B (AS-B) inhibitors by reversed-phase high performance liquid chromatography (RP-HPLC) has been established. The contents of asparagines produced in the reaction system can be analyzed by HPLC after the derivatization with 1-fluoro-2,4-dinitrobenzene (DNFB) and used to calculate the total activity of AS-B. The sample was separated on an Agilent C18 column (250 mm x 4.6 mm, 5 microm) at the temperature of 30 degrees C with the elution of 50 mmol/L sodium acetate buffer (pH 6.2)-acetonitrile (15:85, v/v) as mobile phase at a flow rate of 1.0 mL/min. The detection wavelength was set at 365 nm. The enzyme reaction system consisted of 100 mmol/L Tris (tris(hydroxymethyl)aminomehane)-HCl buffer (pH 8.0), 100 mmol/L NaCl, 10 mmol/L MgCl2, 5 mmol/L adenosine triphosphate (ATP), 10 mmol/L L-aspartate, 10 mmol/L L-glutamine and 2 microg recombinant soybean AS-B (1 mL of the total volume), then mixed for 1 min and incubated for 15 min at 37 degrees C. After quenching with ethanol and centrifugation, the supernatant was derivatized by DNFB and then separated by HPLC. The amino acids in the reaction system were baseline separated within 6 min. The quantitative analysis of AS-B inhibition was performed by determining its dynamic parameters. The inhibitor L-glutamic acid gamma-methyl ester was used in the enzyme reaction system to test this method and its inhibition constant obtained was close to the literature value. The established method is fast, accurate, sensitive and suitable for high throughput screening AS-B inhibitors.