Colon cancer is one of most common types of cancer and the second cause of death worldwide. Colon- specific drug delivery systems (CDDS) are carried out to reduce systemic side effects and improving protein-drugs' oral bioavailability. Bee Venom is a protein that inhibits growth of cancer cells. Three different oral Bee Venom (BV)-chitosan microspheres formulations were prepared and evaluated using different BV: chitosan ratios 1:1, 1:2 and 1:3 for formulations F1, F2 and F3 respectively. Coating of microspheres was done using Eudragit S100 (ES100) by solvent evaporation method. Pre-clinical study was done to determine oral safety dose of BV and LD50 using rat. The prepared microspheres were characterized by percentage yield, degree of swelling, surface morphology using scanning electron microscope (SEM), in-vitro release study and entrapment efficiency. Upon determination of its safety dose on rats; Results showed that the maximum safety oral dose appeared to be 64 mg/kg after pre-clinical investigation. The percentage of drug release study after 12 hours was found to be 82.35, 75.9, and 60.26 % for the formulations F1, F2 & F3, with a percentage yield of 80, 89.1 & 92.35% respectively. The degree of swelling showed the ability of BV-ES100 coated microspheres to resist the pH in upper GIT. The microspheres were spherical shaped with a smooth surface upon scanning with SEM. In conclusion, the results showed that the coated BV-chitosan microspheres are a promising system for colon- specific drug delivery.