One of the most prevalent tumours in the world is colorectal cancer, which is also thought to be the second largest cause of mortality from cancer. Colon cancer can be treated with 5-fluorouracil (5-FU, also known as 5-fluoro-2,4-pyrimidinedione) or in conjunction with chemotherapy treatments. The present study aimed to create a Eudragit S100 coated colon-targeted microbead method to enhance colon targeting of 5-Fluorouracil by enhancing its local action in the colon.A technique called ionotropic gelation was used to create the formulations. Formulations were assessed based on their surface phenomenon, bead shape, entrapment effectiveness, drug loading, and in vitro drug release. Moreover, Eudragit S100 was enteric coated on Calcium alginate beads with an improved core.The particle size of the optimized formulation was 895 μm, drug entrapment efficiency was 89.91%, drug loading was 38.64%, and in vitrorelease was 79.35% after 6 hours in phosphate buffer at pH 6.8. The drug release was 55.12±1.23% after 10 hours following enteric coating of optimized calcium alginate microbeads with Eudragit S100 (10% weight increase). Regression coefficient values for the zero order, first order, and Higuchi models were determined to be 0.9790, 0.7529, and 0.9887, respectively. Based on percent cell viability, the 50 percent inhibitory concentration value found was 3.252.