Abstract: Background and Objective: Celiac disease in genetically susceptible in individuals. It is resulted from T cell intolerance to wheat gluten that is rich with proline residue and are highly resistant to degradation by the proteolytic enzymes secreted by the gastrointestinal tract. The aim of the current study was to purify and identify new prolyl endopeptidase from natural and economical source such as larva midgut of red palm weevil (Rhynchophorus ferrugineus) able to degrade gluten at low gastric pH. Materials and Methods: The prolyl endopeptidase was extracted from larva midgut of red palm weevil (Rhynchophorus ferrugineus) and purified using ammonium sulphate, acetone and trichloroacetic acid. The enzyme activity was tested of hydrolysis efficacy by gluten, gliadin and peptide peptidyl-pNA. Results: The results showed that 80% acetone give high enzyme activity, high Vmax value and low Km value. At 2 mg mL–1, the efficacy to hydrolyze both gluten and gliadin were found higher than digestine, a positive control. The optimum enzyme activity was at pH 5 and 30°C, the stability of enzyme activity was remaining at temperature reached to 50°C and pH 4.0. Conclusion: The results reflect the potential use of the prolyl endopeptidase in the medical and processing of gluten free food applications.