Aims of the study: The purpose of this experimental work was to quantitatively determine the concentrations of nicotine and cotinine and trans-3-hydroxycotinine in biological urine samples from voluntary subjects before and after their visit to a nightclub in which smoking was allowed, by using the GC-MS method. Material and methods: Twenty-two subjects participated in the research, including 7 men and 15 women. Biological urine samples of unknown nicotine and metabolite concentrations were prepared for analysis by the liquid-liquid extraction (LLE) method. For analysis, we used the GC-MS method. Results: All samples were positive for nicotine metabolites. The mean value of nicotine, cotinine and 3-OHK before exposure at a nightclub in non-smokers was 8,197 ng / mL, 28,372 ng / mL, 12,303 ng / mL in women and 11,235 ng / mL, 25,763 ng / mL, 3,673 ng / mL in men. Mean post-exposure values were: 123,373 ng / mL, 56,544 ng / mL, 41,501 ng / mL in women and 21,366 ng / mL, 65,234 ng / mL, 14,941 ng / mL in men. Conclusions: Concentrations of nicotine and metabolites in non-smokers before exposure were significantly lower than after exposure. The levels of nicotine and metabolites in smokers are significantly higher than in non-smokers. Considering the length of exposure, the increase in concentrations of nicotine, cotinine, and 3-OHK in non-smoker waiters was higher than in male respondents who stayed in the club as visitors.