Abstract: Anticholinesterase pesticides are widely used, and as a result they are involved in numerous acute and even fatal poisonings. The aim of this study was the development, optimization, and validation of a simple, rapid, specific, and sensitive gas chromatography–mass spectrometry method for the determination of 11 anticholinesterase pesticides (aldicarb, azinphos methyl, carbofuran, chlorpyrifos, dialifos, diazinon, malathion, methamidophos, methidathion, methomyl, and terbufos) in blood. Only 500 μL of blood was used, and the recoveries after liquid–liquid extraction (toluene/chloroform, 4:1, v/v) were more than 65.6%. The calibration curves were linear (R2 ≥ 0.996). Limit of detections and limit of quantifications were found to be between 1.00–10.0 and 3.00–30.0 μg/L, respectively. Accuracy expressed as the %Er was found to be between −11.0 and 7.8%. Precision expressed as the percent relative standard deviation was found to be <9.4%. The developed method can be applied for the investigation of both forensic and clinical cases of accidental or suicidal poisoning with these pesticides.