The present study compares three colon-targeted drug delivery systems; Eudragit S100-coated polymeric tablets, PLGA nanoparticles, and alginate hydrogel microspheres, developed for the controlled release of 5-Fluorouracil (5-FU). Each formulation was prepared and optimized using distinct carriers and evaluated under simulated gastrointestinal (GI) conditions to assess their physicochemical characteristics, release behaviour, and stability. The formulations were characterized for particle size, surface charge, encapsulation efficiency, and swelling index. Morphological analysis confirmed smooth coating in polymeric tablets, spherical uniformity in nanoparticles, and a porous structure in hydrogels. In vitro dissolution studies revealed minimal drug release in gastric conditions (≤2% at pH 1.2) and sustained release at colonic pH (7.4). PLGA nanoparticles showed the most controlled release profile, achieving 92.1 ± 2.4% cumulative release at 24 hours, compared with 100.0 ± 3.1% for polymeric tablets and 85.4 ± 2.1% for hydrogels. Kinetic modeling indicated that all systems followed diffusion-dominated release, with nanoparticles best fitting the Higuchi model (R² = 0.981). Stability studies confirmed nanoparticle integrity under prolonged acidic and neutral exposure, while hydrogels exhibited partial deformation. Overall performance analysis identified PLGA nanoparticles as the most efficient system, demonstrating superior acid resistance, encapsulation efficiency, and colon-specific release. These findings suggest that nanoparticle-based carriers offer significant potential for achieving predictable, site-specific, and sustained drug delivery to the colon.