The severe effects of alcohols on humans trigger the continuous research on the alcohols level measurement in biological fluids. The officially established technique is Headspace Gas Chromatography (HS-GC), while breathalyzers are commonly used by police on the road. However, they all exhibit drawbacks; HS-GC is expensive and labor-intensive, while the precision of breathalyzers is controversial. In the present study, a novel method was developed, for ethanol and methanol detection and quantification in human urine, saliva and blood serum, based on Raman spectroscopy. Biological fluids from healthy adult volunteers were collected, standard solutions of the alcohols in a concentration range from 0.00 μL/mL to 5.00 μL/mL were prepared and analysed using an air-tight and small volume sample carrier. Calibration curves for each binary system (alcohol – biological fluid) were created. Ethanol calculated detectable concentrations were below permissible limits for all biological fluids. In the case of methanol, the limits were not as satisfactory, but lower than intoxication level, due to the difficult spectral discrimination. For both alcohols, the lowest detection limits were recorded for saliva. All detection limits were verified by visual inspection of the spectra. The proposed quantitative method was validated in all cases regarding their specificity, working range, accuracy, precision and sensitivity.