Introduction: Smokeless Tobacco contains considerable nicotine much more than is contained in smoking tobacco. Smokeless Tobacco contains 28 carcinogenic agents, including nitrites and alkylating agents causing oral cancer. Ethanol is the principal ingredient of alcoholic beverages Acetaldehyde, a metabolite of ethanol play an important role in ethanol toxicity by cross linking the DNA and thus leading to DNA damage. Comet Assay was performed to assess DNA damage among users of Smokeless Tobacco and Alcohol. Method: Buccal epithelial cells were taken from 25 users of Smokeless Tobacco (Khaini) 25 users of Alcohol, 25 users of Khaini and Alcohol and 25 control subjects and Comet Assay was performed to assess possible DNA damage. Result: Highest increase in the comet tail length implicated DNA damage in users of Smokeless Tobacco and Alcohol, both as compared to the control subjects. Conclusion: This study demonstrated DNA damage among users of Alcohol, Smokeless Tobacco leading to the development of oral cancer.