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Objective: This study evaluated the sealing ability of different biomaterials as intra-orifice barriers in the internal bleaching of discolored teeth with the walking bleaching technique. The release of hydroxyl ions from the bleaching materials can cause cervical root resorption, making it necessary to use intra-orifice barrier materials to prevent this issue. Materials and methods: In the current study, the high-performance liquid chromatography (HPLC) method was used to measure the released hydroxyl ions. The study included 90 single-rooted and single-canal premolars, which were divided into four groups based on the intra-orifice barrier materials used (mineral trioxide aggregate [MTA], calcium-enriched mixture [CEM], Biodentine, and MTA+PG) and the type of bleaching material (sodium perborate + water or sodium perborate + hydrogen peroxide 30%). Two control groups were also considered in this study: a positive control group, where sodium perborate and hydrogen peroxide were placed inside the pulp chamber without any intra-orifice barriers; and a negative control group, where no bleaching agent or surgical obstruction was used, and the root surface was covered with wax up to the cemento-enamel junction (CEJ) level. Results: The results showed that there was a significant difference in the concentration of hydroxyl ions released among the studied groups. The amount of hydroxyl ion released was highest in the positive control group and lowest in the CEM group. Among the intra-orifice barrier materials used, CEM cement was found to be the most appropriate material for use in the step-by-step internal bleaching method. Conclusions: The study highlights the importance of using appropriate intra-orifice barrier materials to prevent root cervical resorption in internal bleaching procedures.