Background: Although nicotine dependence plays a role as a main barrier for smoking cessation, there is still a lack of solid evidence on the validity of biomarkers to determine nicotine dependence in clinical settings. This study aimed to investigate whether urinary cotinine levels could reflect the severity of nicotine dependence in active smokers. Materials and Methods: Data regarding general characteristics and smoking status was collected using a self-administered smoking questionnaire. The Fagerstr$ddot{o}$m test for nicotine dependence (FTND) was used to determine nicotine dependence of the participants, and a total of 381 participants were classified into 3 groups of nicotine dependence: low (n=205, 53.8%), moderate (n=127, 33.3%), and high dependence groups (n=49, 12.9%). Stepwise multiple linear regression model and receiver operating characteristic (ROC) curves analyses were used to determine the validity of urinary cotinine for high nicotine dependence. Results: In correlation analysis, urinary cotinine levels increased with FTND score (r=0.567, P<0.001). ROC curves analysis showed that urinary cotinine levels predicted the high-dependence group with reasonable accuracy (optimal cut-off value=1,000 ng/mL; AUC=0.82; P<0.001; sensitivity=71.4%; specificity=74.4%). In stepwise multiple regression analysis, the total smoking period (${eta}$=0.042, P=0.001) and urinary cotinine levels (${eta}$=0.234, P<0.001) were positively associated with nicotine dependence, whereas an inverse association was observed between highest education levels (>16 years) and nicotine dependence (${eta}$=-0.573, P=0.034). Conclusions: The results of this study support the validity of using urinary cotinine levels for assessment of nicotine dependence in active smokers.