The limitations of clinical chemotherapy are credited primarily to drug resistance. Effective development and screening of new drugs require appropriate in vitro tumor models that resemble the in vivo situation to evaluate drug efficiency and to decrease the use of experimental animals. 3D in vitro model systems that are able to mimic in vivo microenvironments are now highly sought after in cancer research. Here, the characteristics of breast cancer cell line MDA-MB-231 cells on 3D, and 2D Antheraea mylitta silk matrices and tissue culture plates are compared. After long term culture of breast cancer cells in the silk scaffold, the engineered tumor construct shows different zones of cell proliferation, such as an avascular tumor. Silk fibroin matrix 3D tumor models are studied for the evaluation of various anticancer drugs. The cytotoxic effects of three different drugs (Paclitaxel, Celecoxib, and ZD6474) at different concentrations are evaluated for MDA-MB-231 grown on 2D films as well as on a 3D fibroin scaffold. Higher drug concentrations are required to achieve a comparable reduction in cell viability and invasive potential in 3D culture. Combinatorial treatment of drugs at IC50 concentrations result in up to 84% death of cancer cells. The results indicate that 3D in vitro tumor models may be better systems to evaluate cancer treatment strategies.