This study presents a headspace gas chromatography method with flame ionization detection (GC-FID) for the determination of ethanol and methanol content in biological fluids such as blood, urine, and saliva, emphasizing matrix effects onto headspace analytical results. In this context, a simultaneous procedure was established and validated, and it was revealed that headspace GC for methanol and ethanol determination in all routinely available biological fluids is applicable as a reference method and also in routine diagnostics and monitoring. GC was performed with a run time of 6 min. The method showed linearity in the range of 50-400 mg/dL both for methanol and ethanol (r(2) = 0.998 and r(2) = 0.999, respectively) in all specimens with a detection and quantification limits less than 1.5 mg/dL and 2 mg/dL, respectively, as well as with good repeatability. Relative standard deviation and percent bias were calculated for interday and intraday accuracy and precision at three concentrations (low 50 mg/dL, medium 200 mg/dL, and high 400 mg/dL) for each biological fluid. A recovery rate of over 90% was obtained. Due to matrix effects of specimens, slight variations in validation data were presented and compared. The method and the obtained results suggest that the use of headspace GC could be extended from confirmatory analyses to routine application in toxicology laboratories.'