In the present study, A fluorescent imaging-based high-throughput screening method was developed for identifying anti-migratory compounds with 96-well Transwell plates.  The correlation, precision and stability of this method were examined and the incubation time of dye Hoechst 33342 in addition to migration time was optimized.  In addition, The inhibitory activity of anti-cancer drug paclitaxel on tumor cell migration was assayed and an IC₅₀ value of 0.717 μmol·L⁻¹ was obtained.  Using this method, 24 components from Rhizoma Alismatis were screened and one component with anti-migration activity was found.  These results show that the new proposed method with good precision, stability and linear range has the potential to assay the inhibitory activity of anticancer compounds.