Abstract: Background: An endodontic treatment aims to recover and maintain dental elements, which have pulpal or endodontic periapical pathology, thus eliminating the microorganisms to ensure an acceptable apical seal. Objectives: The three different conditions using naked vision, loupes and Dental operating Microscope were compared here to test the sealing ability of biodentine using glucose penetration method. Methods: Ninty mandibular bicuspids were selected that were further divided into three groups (n=30) for which canal were prepared using F2 ProTaper Universal (Dentsply Maillefer, Switzerland) and a retrograde cavity is prepared in each of the extracted single rooted tooth using proultra surgical tip (Dentsply, USA) 3mm short of apex . Cavity was then filled with biodentine condensed against plugger placed inside the canal. All the procedure were confirmed under unaided vision/ naked vision, under dental loupes (IDS Denmed 3.3X). and under Dental operating Microscope (Carl zeiss 12.5X) for each group. All the samples were then analyzed with a spectrophotometer at a suitable wavelength. The (ANOVA) test of comparison was analysed for multiple groups preceded by the post hoc pair-wise comparison test. Results: The filling of biodentine is observed under dental operating microscope that showed better sealing properties when compared to the unaided vision and dental loupes, thus proving statistically significant. The glucose leakage evaluated under working conditions namely unaided vision, dental loupes and Dental operating Microscope were 0.236, 0.111 and 0.055 respectively. Conclusion: The restoration done under Dental operating Microscope showed superior sealing properties when compared to the other working conditions respectively.