Verification of the results of questionnaire studies of exposure to tobacco smoke require determination of the biomarkers in body fluids. For performing such measurements the fast and simple method is needed. The aim of this study was to estimate usefulness of enzyme-linked immunosorbent assay (ELISA) in routine cotinine determination in urine of active smokers and compare it with reference high performance liquid chromatography method (HPLC). In the study participated 15 non smoking and 15 smoking women. In the urine of tese women cotinine concentration of cotinine was measured by the means of HPLC and ELISA methods. The ELISA method, used in quantity measurement of cotinine in urine, is of great accuracy, sensitivity and specificity which were proved by comparison with high performance liquid chromatography (HPLC) reference method. Number of false positive and false negative results obtained by ELISA method did not exceed 10%. A high correlation coefficient, r=0.9056, between results of determining cotinine in urine by means of ELISA and HPLC methods, confirmed the utility of ELISA method to estimate the tobacco smoke exposure. The differences in cotinine concentration values obtained by ELISA and HPLC methods did not depend on cotinine concentrations in the urine samples. Although numerous advantages of ELISA method used to detect and determine cotinine, it should be taken into consideration that results might be overestimated and cross-reactivity with other xenobiotics present in urine must be concern.