This study aimed to optimize MES-nanoparticles using Box Behnken Design (BBD) and investigate its in vivo antioxidant potential in colon drug targeting. The formulation was prepared using oil/water (O/W) emulsion solvent evaporation technique for time dependent colonic delivery. The optimal formulation with the following parameters composition was selected: polymer concentration (% w/w) (A) = 0.63, surfactant concentration (% w/w) (B) = 0.71, sonication duration (min) (C) = 6. The outcomes showed that ethyl cellulose nanoparticle containing mesalamine has particles size of 142 ± 2.8 nm, zeta potential of -24.8 ± 2.3 mV, % EE of 87.9 ± 1.6%, and PDI of 0.226 ± 0.15. Scanning electron microscopy revealed nanoparticles has a uniform and spherical shape. The in-vitro release data disclosed that the ethyl cellulose nanoparticles containing mesalamine showed bursts release of 52±1.6% in simulated stomach media within 2 hours, followed by a steady release of 93±2.9% in simulated intestinal fluid that lasted for 48 hours. The mesalamine release from nanoparticle best match with the Korsmeyer-Peppas model (R2 = 0.962) and it followed fickian diffusion case I release mechanism. The formulation stability over six-months at 25 ± 2 °C with 65 ± 5% relative humidity, and 40 ± 2 °C with 75 ± 5% relative humidity showed no significant changes changes in colour, entrapment efficiency, particle sizes and zeta potential. As per in vivo results, MES-NP effectively increased GSH, SOD level and reduces the LPO level as compared to other treatment groups. The findings hold promise that the developed formulation can suitably give in ulcerative colitis.