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Hydrogels, because of their adaptive properties and biocompatibility, have become promising platforms for controlled drug delivery. In this study, protein release kinetics from hyaluronic acid (HA)-based hydrogels incorporating fibronectin and collagen were examined to elucidate the influence of the hydrogel composition on the release profiles of proinsulin variants. HA- collagen-fibronectin hydrogels were synthesized according to a standardized protocol, with variations in collagen and fibronectin concentrations. Three proinsulin variants, F25D, F25D- K3, and F25D-K12, were used along with the hydrogels to conduct protein release assays over 24 hours using fluorescence endpoint measurements. Distinct release profiles were observed for each proinsulin variant. For F25D, hydrogels containing fibronectin at 0.125 mg/mL and collagen at 1.0 mg/mL exhibited the highest protein release, with a significant increase observed from 12 to 24 hours. Collagen, particularly at 1.0 mg/mL, showed the most considerable protein release across all variants, while fibronectin demonstrated comparatively lower release rates, especially at 0.25 mg/mL. The findings underscore the importance of hydrogel composition in modulating protein release kinetics. For instance, collagen emerged as a critical component in protein release, whereas fibronectin exhibited lower release rates. The results provide valuable insights for hydrogel-based drug delivery systems, emphasizing optimizing hydrogel compositions to achieve desired and realistic release profiles for real-life therapeutic applications. Further research might explore factors influencing protein release dynamics, facilitating more effective drug delivery strategies.