Background:Dentin Hypersensitivity is a common dental problem affecting a huge section of every population that doesn't have a permanent treatment yet. Aim: The purpose of this study is to compare the effect of Diode laser (940nm) and Nano-desensitizing agent on the degree of tubular occlusion compared to the conventional fluoride varnish for the treatment of dentin hypersensitivity. Materials and methods: Twenty dentin specimens were prepared from extracted human premolars. Dentin specimens will be randomly allocated in 4 groups (n=5) according to desensitizing agent used: D₁ (Diode Laser (940nm), D₂ (Nano-hydroxyapatite), D₃ (Diode Laser + Nano-hydroxyapatite) and D₄ (Fluoride). The specimens will be treated with EDTA for 2 mins to open dentinal tubules simulating Dentin Hypersensitivity. Each specimen will be evaluated at 3 different times: T₀ (before material application), T₁ (after material application) and T₂ (after acid attack) using Environmental Scanning Electron Microscope and Image Analysis software to detect degree of occlusion of dentinal tubules. Results: Group D₃ showed statistically significant tubular occlusion percentagecompared to the other groups except group D₁. After acid attack group D₃ were the only group that showed non-statistically significant decrease in tubular occlusion percentage denoting the best resistance to acid attack, while other groups were drastically affected by the acid attack. Conclusion: Combining DL (940nm) and Nano-hydroxyapatite could be a successful treatment option for Dentin Hypersensitivity, due to its high degree of tubular occlusion effect and sustainability in the acidic environment.