Background: This study was conducted to evaluate serum nicotine levels in tobacco users. Material and methods: 100 subjects were recruited in all which were divided into two groups of 50 subjects each. Group A comprised of 50 tobacco users while Group B was the control group with 50 non users. For each participant, a questionnaire assessed tobacco use. Serum nicotine levels were measured. Results: Mean cotinine levels were 93.26 ± 16.73 and 26.54 ± 9.2 ng/ml among tobacco smokers and non-smokers, respectively. Cotinine ≥15 ng/ml was measured in 81% of smokers and in 23% of non-smokers. Conclusion: From the results, it was concluded that serum nicotine levels among tobacco users were higher in comparison to non-users.