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Gluten and gliadin, which are present in wheat, barley, and rye, can cause celiac disease that causes intestinal inflammation and nutrient loss in those who are genetically predisposed. Currently, the only effective treatment is strict lifelong gluten abstinence. This study aimed to evaluate the efficiency of protease enzymes extracted from germinated barley in degrading gluten proteins, with a particular focus on their application in the production of rice biscuits suitable for individuals with gluten sensitivity. Protease enzymes were extracted and partial purified from germinated barley and characterized for their activity and stability at different temperatures and pH levels. The enzymatic hydrolysis of gluten and gliadin proteins was assessed using protein determination, residual gluten quantification and docking study of barley protease and the peptide fragment of gluten and gliadin. The results showed that protease was partially purified by using 60% saturated crude extract salted-out by ammonium sulphate, followed by dialysis. The stability of protease enzyme was by evaluating the enzymatic activity at different pH values and temperature. The results showed that germination significantly enhanced protease activity compared to ungerminated barley, with optimum activity observed at 35–45 °C and pH 7.0. Treatment with the extracted protease caused extensive degradation of gluten (14.75 %). Incorporation of the germinated barley protease into rice biscuit formulations improved dough handling, texture, and sensory properties while reducing gluten levels to a range suitable for gluten-sensitive consumers. These findings highlight the potential of germinated barley protease as a natural, plant-based enzyme source for producing gluten-reduced rice biscuits, offering a promising strategy for developing functional bakery products tailored for individuals affected by celiac disease.