The resistance to Methotrexate by a number of cancer cells including breast cancer cell line MDA-MB-231 due to poor permeability renders it less effective as an anticancer agent for these cells. Prodrug of Methotrexate (Pro-MTX) was designed as a substrate of prolidase which is specific for imido bond of dipeptide containing proline and expected to penetrate MDA-MB-231 cells more efficiently and be activated by higher prolidase activity in MDA-MB-231 cells. The prodrug was synthesized by solid phase peptide synthesis method and examined as a substrate of pure prolidase as well as cell homogenate. The cytotoxicity against MDA-MB-231 and non-methotrexate resistant breast cancer cell line, MCF-7 was also examined by XTT assay. The results show Pro-MTX is a substrate of prolidase. It is also shown that the prodrug is able to be converted to parent drug methotrexate in caco-2 and HeLa cell homogenate. When tested with Caco-2 cells, pro-MTX shows weaker cytotoxicity compared with methotrexate. Pro-MTX has estimated GI₅₀ value of 6μM while methotrexate has a GI₅₀ value of 0.8 μM. This is also confirmed by other studies showing methotrexate amino acid prodrugs are generally weaker than the parent drug for their anti-proliferate activity. The results for MCF-7 cells are similar with caco-2 cells. Methotrexate has estimated GI ₅₀ value of 0.9 μM while pro-MTX shows a GI₅₀ value of 5μM. But for methotrexate resistant MDA-MB-231 cells, pro-MTX shows stronger activity than methotrexate. Pro-MTX has estimated GI₅₀ value of 11μM while methotrexate shows a GI₅₀ value of 80μM. The results indicate that the proline prodrug of methotrexate may overcome the resistance of human breast cancer cells in culture.