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Introduction: Tooth hypersensitivity is among the most common patient complaints caused by the response of exposed dentin to external stimuli. No definite treatment has been suggested so far for dentin hypersensitivity (DH). This study aimed to compare the efficacy of the diode laser alone and in combination with Gluma and chromophore in occluding opened dentine tubules and the treatment of DH. Methods: This in vitro study was conducted on 30 extracted human third molars kept in 0.1% thymol solution. The teeth were mounted in transparent acrylic resin and the buccal enamel was cut to expose the outer third of dentin. The samples were then divided into 6 groups of negative control (no smear layer removal), positive control (smear layer removal with 6% citric acid application), 810 nm diode laser irradiation (energy density 55.55 J/cm2, 1 W for 20 seconds in a continuous mode), chromophore (1 mg/mL indocyanine green, ICG) plus diode laser irradiation (1 W, 20 seconds), Gluma plus diode laser irradiation, and Gluma + chromophore + diode laser. Dentinal tubules were evaluated under a scanning electron microscope at x2000 magnification. The mean percentage of the obstruction of dentinal tubules was reported as mean and standard deviation. Considering the normal distribution of the data, two-way ANOVA was applied to compare the efficacy of treatments, and an independent-samples t test was used for pairwise comparisons at P<0.05 level of significance. Results: The highest mean percentage of the obstruction of dentinal tubules was noted in the diode laser/chromophore/Gluma group (65.68 ± 12.31%) while the lowest value was noted in the diode laser/Gluma group (24.33 ± 5.90%). Pairwise comparisons revealed significant differences between all groups (P < 0.05) except for the difference between the laser/Gluma/chromophore and laser/ chromophore groups (P = 0.20). Conclusion: It seems that chromophore increases the efficacy of the diode laser for the obstruction of dentinal tubules. [ABSTRACT FROM AUTHOR]