Aim: This study aimed to design, develop, and evaluate polymeric mucoadhesive microspheres loaded with ondansetron hydrochloride for intranasal delivery. The objective was to bypass first-pass metabolism in the liver, enhance bioavailability, and extend the residence time of the drug. Methodology: Microspheres were formulated using the solvent evaporation technique with carbopol 940 and HPMC K15M as mucoadhesive polymers, alongside ethyl cellulose as a film-forming polymer. The study evaluated the impact of formulation variables, such as polymer-to-polymer ratio and stirring rate, on the microsphere characteristics. Comprehensive characterization included assessments of drug-polymer interactions, entrapment efficiency, drug loading, swelling properties, particle size analysis, thermal behavior, morphology, in-vitro mucoadhesion, ex-vivo drug permeation, in-vitro drug release, histopathology, and release kinetics. Results: The microspheres demonstrated favorable characteristics, including satisfactory entrapment efficiency, controlled drug release, and excellent mucoadhesive properties. Particle size analysis confirmed uniformity, while morphological studies revealed a spherical structure. In-vitro and ex-vivo evaluations indicated effective drug permeation and sustained release. The histopathological findings confirmed the safety of the formulation, and release kinetics followed a controlled release profile. Conclusion: The results establish that ondansetron hydrochloride-loaded mucoadhesive microspheres, prepared using the solvent evaporation method, offer a promising approach for intranasal drug delivery. This formulation strategy effectively enhances bioavailability, provides sustained drug release, and has the potential for future advancements in nasal drug delivery systems.