The present study is aim at the development of PLGA based nanoparticles of Doxorubicin for anti breast cancer activity by in silico and in vitro cell line using MCF-7. Nano formulated Doxorubicin was standardized with various parameters such as drug excipient interaction, particle size, shape, surface morphology, polydispersity index, zeta potential, drug content, entrapment efficiency etc. Thereafter, tested as a pure compound, 99 percent of medications are released right away. Doxorubicin was prepared as PLGA nanoparticles with NP1 and NP2, an extended release was noted, indicating the drug release over a period of 2 days and NP3 and NP4 over a period of 4 days. Thereafter, molecular docking study was performed for Doxorubicin with PDB protein enzyme 3ERT and 4OAR. Finally, in vitro breast cancer activity was performed using MCF-7 cell line. Overall result concluded that doxorubicin showed binding energy of -8.5 with 3ERT protein and -8.8 with 4OAR protein complex. The best performing formulation was NPF3 which constituted doxorubicin nanoparticles of appropriate size and demonstrated benefits of controlled drug release over free doxorubicin and other formulations. Finally, in vitro MCF-7 cell line study for breast cancer was performed with best formulation which showed remarkable inhibition of cancer growth by measured dose dependent decreased in Glutathione level and increased in lipid peroxidation level.