Introduction: Samples used for post-mortem alcohol analysis are femoral vein blood samples and urine. Recently, the Croatian law mandates simultaneous sampling of aqueous humour as well. Exceptionally, when it is not possible to collect samples mentioned above, the alcohol concentration can be determined from the femoral muscle distillate by formula calculation which reflects blood ethanol concentration. Objective: The aim of this study is to determine whether the ethanol concentration in the femoral muscle distillate can be used to estimate blood alcohol concentration. Study Design: Cross-sectional study. Methods and materials: 16 femoral vein blood, aqueous humour and femoral muscle samples were collected during the autopsy at the Clinical Institute for Pathology and Forensic Medicine of the Clinical Hospital Centre in Osijek and were used for the analysis. Samples were prepared by deproteinization with trichloroacetic acid; femoral muscle samples were prepared by distillation and enzyme essay method on a VIVA E analyser (Siemens Healthcare Diagnostics Inc., Newark, USA). Results: Paired difference testing of concentration values from different samples using the Wilcoxon test revealed a statistically significant difference between blood and muscle (P<0.001) samples, whereas the difference between aqueous humour and blood samples was not present (P=0.860). The Passing-Bablok regression analysis indicates that the alcohol concentrations measured in the aqueous humour and in the blood are consistent (Y=0.160+0.953x). Paired values of the blood and muscle samples findings indicate a constant but not proportional difference (Y=-0.137+0.951 x). Conclusion: The results of this study indicate a correlation between the ethanol concentrations levels obtained from aqueous humour and blood analysis, while the ethanol concentrations obtained from the femoral muscle analysis are inconsistent with the blood alcohol concentrations. When interpreting the findings obtained from all biological samples, one must always approach the toxicological evaluation with extreme caution, taking into account the sample choice, methods used and the potential sources of analytical errors.