Objective: The present study aims to prepare and improve polymeric nanoparticles for rutin andberberine site-specific delivery in cancer treatment. Eudragit, a pH-sensitive polymer used in theformulation of the nanoparticles, was used to achieve site-specific distribution. Materials and methods: Nanoparticles were prepared by using ionic gelation method. For formulationoptimization and validation, the central composite design has been used. SEMmicrographs,nanoparticlesizeanalysis,zetapotentialanalysisbyDLS,encapsulationeffectiveness,in-vitrodrugreleaseprofile,andstabilitywereallthe parameters evaluatedfortheoptimized formulation. Results: The characterization results showed that the nanoparticles' surfaces weresmooth, and the particle sizewas determinedtobe101.4nm. Zetapotentialwasdeterminedtobe -0.98±0.05mV and encapsulation efficiency to be 92.96% for rutin and 96.96 % for berberine,respectively.Rutinandberberine-loadednanoparticles (RB-NPs) werefoundtobecytotoxicwhentested in-vitro on the EAC (Ehrlich-Lettre Ascites-E) cell line. Rutin and berberine-loaded NPSwere shown to have a percentage cell viability of 83.09 % and 6.25 g/ml, respectively.