Objective: Although medicine students express positive attitudes toward providing lifestyle counseling, they require more instruction in many areas of health behavior in order to be helpful to their patients. The presented study included the students' questionnaires analysis regarding their lifestyle and exposure to tobacco smoke. The aim of this study was to examine students' exposure to chosen xenobiotics by determination of selected biomarkers in urine samples, which underlay the basis for exposure assessment towards tobacco smoke. Materials and Methods: The investigated group consisted of first- and second-year medicine students from the Silesian Medical University (N = 133). Data obtained from a questionnaire survey was compared with the results of chosen biomarkers determined in urine samples. The analyses of the main nicotine metabolites were carried out firstly with use of ELISA, followed by the TLC technique with densitometry. Results: According to questionnaires, every third student examined was exposed to passive smoking. The mean concentration of the main nicotine metabolites determined by ELISA in urine samples of smoking students was 1293.52±396.70 μg/g creatinine. The results of the TLC analysis in the group of smoking students were as follows: for cotinine - 523.10±68.10 μg/g creatinine and for trans-3'-hydroxycotinine - 653.81±62.30 μg/g creatinine. Conclusions: Medicine students, regardless of their area of study, are a highly-exposed part of the population to tobacco smoke, not only actively but also passively. Tobacco smoke exposure can be assessed by ELISA as a screening method as well as by more specific TLC technique with densitometry.