Abstract: AIMIn order to find novel inhibitors of influenza virus neuraminidase (NA), an assay method of neuraminidase activity was established for high throughput screening. METHODSThe strain A (Yuefang 72-243 A and Jifang 90-15 A) and B (Sichuan 2000-38 B) influenza viruses were used as source of neuraminidase and the activity of neuraminidase was measured by fluorometric method. The reaction system of neuraminidase with substrate, 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA) was optimized by changing the conditions such as concentrations of neuraminidase, substrate, Ca²⁺, reactive system pH and temperature. At last, the inhibitory effects of 1 200 samples (including compounds and extracts from plants) were screened by the fluorometric assay. RESULTS The optimized neuraminidase reaction system contains enzyme, MUNANA 20 μmol·L^-1, Ca²⁺ 4 mmol·L^-1, at pH 3.5 and 37℃. The Michaelis and Menten constants (Km) of influenza virus A (Yuefang 72-243 and Jifang 90-15) and B (Sichuan 2000-38) are (5.9±1.9), (5.2±0.5) and (4.9±1.2) μmol·L^-1 respectively. Among 1 200 samples, there were about 1% showed potential inhibitory effects on influenza virus neuraminidase. CONCLUSION The temperature, pH and concentrations of substrate and ions are very important factors affecting the influenza virus neuraminidase activity in vitro. The fluorometric assay of neuraminidase was performed by automatic laboratory station to screen inhibitors of influenza virus neuraminidase in vitro. It can also be used to study the enzyme inhibition kinetics.