A gas chromatography with flame ionization detection method (GC-FID) with direct injection, using a capillary column, was validated to determine ethanol, acetaldehyde, methanol, and acetone in different human matrices, such as whole blood, vitreous humour, and urine, with clinical and forensic interest. This method was also employed to quantify these compounds in cell culture medium, thus being useful in basic research. A good peak resolution was achieved, with linear correlation between concentration and peak areas for all the compounds in all the matrices. The inter- and intra-day precisions of the method were always under 15% and 10%, respectively. The accuracy of the method, calculated as the percentage of the target concentration, was within the acceptable limits. The obtained limits of detection were below 0.85 mg/L for acetaldehyde and below 0.75 mg/L for the other considered compounds. The small injection volume and the high split ratios applied, allied to the high performance of the GC column, resulted in very good peak resolution and high sensitivities. This method is easy to perform, making it suitable for the routine of clinical biochemistry and forensic laboratories.