Natural products provide a new opportunity for the discovery of neuraminidase (NA) inhibitors. In this study, an affinity ultrafiltration (AUF) coupled with HPLC-MS/MS method was firstly developed and optimized for screening of NA inhibitors from natural products. The critical factors influencing the interaction of enzyme-ligand (including sample concentration, enzyme concentration, incubation time and temperature, pH of the buffer, and dissociation solvents and time) were investigated and optimized by a one-factor-at-a-time design. The method was then applied to discover NA inhibitory compounds in stems and leaves of Baphicacanthus cusia. As a result, five active alkaloids were screened out and identified as 2,4(1H,3H)-quinazolinedione (1), 4(3H)-quina-zolinone (2), 2(3H)-benzoxazolone (3), tryptanthrin (4), and indirubin (5) through analysis of their DAD pro-files, MS/MS fragments, and comparison with reference substances. These active compounds were further evaluated for their NA inhibitory activity using a fluorescence-based NA inhibition assay. The result from the fluorescent assay revealed that all the five compounds (1-5) showed pronounced NA inhibitory activities with IC50 values of 98.98, 64.69, 40.16, 69.44, and 144.73 mu M, respectively. Finally, molecular docking of these five alkaloids with NA showed that hydrogen bond and pi-cation interactions dominated within the binding sites with binding energies ranging between-5.7 to-7.9 kcal/mol, which was supported by the results of the AUF and the fluorescence-based enzyme assay. The developed AUF method is simple and efficient for screening potential NA inhibitors from stems and leaves of B. cusia.