Melatonin has shown great promise as a beneficial molecule in bone regeneration because of its antioxidant, antiinflammatory, and bone-forming properties. However, its short biological half-life limits its effectiveness when given systemically. This study aimed to evaluate how melatonin is released in vitro from hyaluronic acid and hydroxyapatiteinfused melatonin (HHIM) sheets, which were developed to deliver treatment locally. HHIM sheets were made using a composite of hyaluronic acid and hydroxyapatite that contained melatonin. Control sheets without melatonin were also prepared for comparison. We measured drug release using a dialysis diffusion method in phosphate-buffered saline (pH 7.4) at 37 ± 0.5°C with gentle agitation. At set times over 72 hours, we took samples and analyzed them using ultravioletvisible spectrophotometry to calculate the total drug release. The HHIM sheets showed a steady and controlled release profile. There was a mild burst of release in the early hours, followed by a gradual and extended release phase, reaching about 94% total release at 72 hours. No release was detected in the control sheets. This sustained release behavior is linked to the hydrated network of hyaluronic acid and the structural role of hydroxyapatite, which together control the release of melatonin. Considering melatonin’s important role during the inflammatory and proliferative stages of bone healing, keeping it available locally for a longer time could improve the activity of bone-forming cells, the formation of the extracellular matrix, and mineralization. Despite the limitations of this in vitro study, HHIM sheets show promise as a localized drug delivery system for bone regeneration, indicating a need for further in vivo research.