Anticancer peptides development can be sped up by using suitable in vitro tumor models. In cancer research, three-dimensional (3D) in vitro models are bridging the gap between two-dimensional cultures (2D) and in vivo murine models. The 2D cultures fail to supply an appropriate microenvironment for screening new anticancer agents. Thus to imitate the in vivo characteristics of cancer in humans and assessing the anticancer drugs efficacy prior to in vivo studies, a 3D model needs to be used. We used previously purified and characterized pro-apoptotic cyclic octapeptide, cyclosaplin in the present study. Here, we evaluated the efficacy of cyclosaplin on 3D silk tumor model using MDA-MB-231 cells in vitro. MDA-MB-231 cells showed good proliferation rate in 3D silk scaffolds using the available pores forming clusters. In 3D constructs, the proliferation rate, glucose consumed, LDH, and MMP-9 activity of human breast cancer cells (MDA-MB-231) were higher compared to 2D. The anti-proliferative effect of the peptide on 3D silk tumor model was monitored by MTT assay, with 2D culture as control. In 3D culture, a high concentration of cyclosaplin was essential to attain 50% inhibition in MDA-MB-231 cells. In 3D constructs, MMP-9 activity reduced significantly in the cyclosaplin treated MDA-MB-231 cells. Microscopic analysis displayed the cellular aggregates formation distributed throughout the scaffolds. The cyclosaplin induced MDA-MB-231 cells decreased in number and size. Overall, the results highlight the promising role of cyclosaplin in cancer therapeutics.