A rapid, accurate and specific proton nuclear magnetic resonance (1H NMR) spectroscopic method is developed to determine ethanol in blood, known as the blood alcohol concentration (BAC). The limits of detection and quantification are 0.02 g/L and 0.07 g/L, respectively. The 1H NMR spectra show linearity for whole blood and serum samples of a concentration range of 0.00–3.00 g/L (R2 > 0.9995). The 1H NMR method is applied and validated for whole blood as the sample media. Real driving under influence case samples are analyzed with the reference enzyme-based alcohol dehydrogenase and headspace gas chromatography techniques by the Forensic Medicine in Bonn. The reference results are compared with the 1H NMR spectroscopic results. The validation and comparison indicate that 1H NMR is suitable for the quantification of BAC in whole blood. This technique has the advantages of automated analysis with good measurement precision and fast sample throughput. A drop of blood (V = 20 μL) is adequate for an analysis leading to a possible simplification of the sample collection. Due to the non-destructive method, follow-up examinations by 1H NMR spectroscopy or DNA determinations by different techniques (PCR, in situ hybridization) are possible in resolving legal disputes.