Aims. To establish the gender-specific cut-off value of phosphatidylethanol (PEth) concentration in the blood for detecting alcohol dependence. Methodology. Using the method of high-performance liquid chromatography - tandem mass spectrometry (HPLC - MS), the concentration of PEth in the blood of 127 alcohol-dependent men and 25 alcohol-dependent women undergoing hospital treatment was determined. To assess the diagnostic accuracy and to establish the gender-specific cut-off values of PEth concentration, a ROC (receiver operating characteristic) analysis was performed. Statistical data processing was carried out using the Statistica 10.0 program. Results. ROC analysis identified a cut-off value of 495 ng/ml for men and 385 ng/ml for women, above which the likelihood of alcohol dependence was high. At this cut-off value, the model demonstrated strong performance with sensitivity of 99% and 100%, specificity of 96% and 98%, PPV of 98% and 96%, and NPV of 98% and 100% for men and women respectively. Conclusion. The results of the study indicate the high diagnostic reliability of PEth as a biochemical marker of alcohol dependence. Gender-specific cut-off values of PEth should be taken into account when using this biochemical marker in the detection of alcohol dependence.