The present study evaluates the suitability of headspace gas chromatography (GC) with a capillary column as a method for determining the ethanol content in different biological fluids. This procedure allows the use of headspace GC not only as a reference method but also in routine diagnostics and monitoring work. The recent literature reviewed reports no standardized methodology that is at the same time suitable for ethanol determination in all routinely available biological fluids (blood, serum, plasma, urine, and saliva). The proposed procedure seems to be a good solution to the problem. The reproducibility study for the biological fluids tested resulted in coefficients of variation that ranged from 0.8 to 2.9% and recoveries that averaged 99%. Linearity was verified in the range of 0.01-20 g/L of ethanol in aqueous solutions. Sensitivity was determined to be 0.01 g/L. Ethanol measurement by this method is easy, simple, and highly reliable, and only a small sample volume (0.1 mL) is required. An internal standard and sample manipulation are not necessary. The obtained results suggest that the use of headspace GC could be extended from confirmatory analyses to routine application in many biomedical fields.