AIM: To establish a high-throughput model for screening anti-tumor agents capable of promoting the polymeriza- tion of tubulin in vitro. METHODS: Tubulin was prepared in different purity for two screening steps. The first step was a high-throughput screening (HTS) for a set of 1500 samples using the GTP-containing tubulin and the end-reading method. The second step was performed on 119 hits from the first screening by a kinetic assay with GTP-lacking tubulin. RESULTS: The HTS for 1500 samples was accomplished in less than 3 h. From the screening, 108 samples were identified with >20 % promotion activity at 10 mg/L. Five of 108 were further confirmed by the kinetic assay using the purified tubulin subsequently. Three of the hit compounds were Epothilone A or its analogs, the other two compounds had new structures with a common pharmacophore for cytotoxic natural products that stabilize microtubules. In an MTT test, the five selected samples from the screening showed a minimal IC50 at0.28±0.06 nmol/Lto Helacells. CONCLUTION: The two-stepscreening methodis a high-throughtput, cost-effective, and efficient approach to identify microtubule-stabilizing agents. AIM: To establish a high-throughput model for screening anti-tumor agents capable of promoting the polymeriza- tion of tubulin in vitro. METHODS: Tubulin was prepared in different purity for two screening steps. The first step was a high-throughput screening ( HTS) for a set of 1500 samples using the GTP-containing tubulin and the end-reading method. The second step was performed on 119 hits from the first screening by a kinetic assay with GTP-lacking tubulin. RESULTS: The HTS for 1500 samples was accomplished in less than 3 h. From the screening, 108 samples were identified with> 20% promotion activity at 10 mg / L. Five of 108 were further confirmed by the kinetic assay using the purified tubulin subsequently. Three of the hit compounds were Epothilone A or its analogs, the other two compounds had new structures with a common pharmacophore for cytotoxic natural products that stabilize microtubules. In an MTT test, the five selected samples from the screening showed a minimal I C50 at 0.28 ± 0.06 nmol / L Helacells. CONCLUTION: The two-stepscreening methodis a high-throughput, cost-effective, and efficient approach to identify microtubule-stabilizing agents.