A new series of 3',4'-bis(3,4,5-trimethoxyphenyl)-4'H-spiro[indene-2,5'-isoxazol]-1(3H)-one derivatives was designed and synthesized. The cytotoxic effects of the synthesized compounds were evaluated on several different human cancer cells. Among them, compound 9e displayed the most potent in vitro antiproliferative activity with IC50 values of 0.07±0.01 µM on T47D cells. Another potent derivative 9h displayed an IC50 value of 0.12±0.07 µM against T47D cells, comparable to that of the positive controls (Colchicine Cisplatin Vincristine Vinblastine Doxorubicin Celecoxib). The structure-activity relationships were discussed and both anti-tubulin and COX-2 inhibitory effects were proposed for the developed compounds.