The Preformulation study conducted for optimizing and validating Bovine Insulin-loaded nanofiber gel formulations involved a comprehensive analysis of various parameters. FTIR spectra confirmed the compatibility of Bovine Insulin with chitosan and sodium alginate, indicating no significant interaction between the drug and polymers. DSC analysis revealed insights into the thermal behavior of Bovine Insulin powder, suggesting a transition at around 51.98°C. X-ray diffraction studies indicated a reduction in peak intensity, hinting at increased solubility and a transition from crystalline to amorphous nature in Bovine Insulin. Determination of thiol groups highlighted the presence of free thiol groups in chitosan and sodium alginate conjugates, with the addition of EDAC enhancing thiol group content. Evaluation of nanofibers demonstrated high drug entrapment efficiency and swelling index, crucial for drug loading and release behavior. Further characterization involved IR spectroscopy, DSC compatibility studies, SEM imaging, TGA analysis, and rheological studies, providing a comprehensive understanding of the formulated nanofiber gels. Moreover, optimization through PEO-loaded nanofiber gel showcased enhanced viscosity, spreadability, extrudability, and drug content uniformity, coupled with favorable drug release profiles, affirming its potential for efficient nasal drug delivery.