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A toxicology laboratory often receives a high number of samples from cases (autopsies or clinical) that may require the quick delivery of trustworthy, accurate results. Thus, there is a great need for a fast and reliable method that is capable of identifying and determining a large number of drugs and drugs of abuse in biological matrices, and especially in blood. In the present study, we describe the development of a fast and simple gas chromatography–mass spectrometry (GC-MS) method for the determination of 41 drugs and drugs of abuse (DOA) in blood. Sample pre-treatment by alkaline liquid–liquid extraction (LLE) was studied through the utilization of different solvents and solvent-to-sample ratios (v/v), which aimed to achieve a greater extraction efficiency and detection sensitivity with a decreased need for large sample volumes. Butyl acetate with a sample-to-solvent ratio of 4:1 (1 mL blood: 0.25 mL butyl acetate) was the most efficient. The method was validated for all analytes, and the evaluation parameters were within the acceptance criteria. The coefficient of determination (R2) was between 0.9934 and 1, the limits of detection (LODs) ranged between 1 ng/mL and 113 ng/mL, and the limits of quantification (LOQs) were between 4 ng/mL and 375 ng/mL for all analytes. The determinations were accurate (accuracy% from 84% to 114%) and precise (RSD% from 0.66% to 14.8% for low concentrations). Deconvolution Reporting Software (DRS) for GC-MS was optimized and applied for data analysis to enhance the identification potential, thereby avoiding false identifications (false positives) and increased productivity. The NIST Automated …