In this study, an accurate and robust gas chromatography/mass spectrometry method was developed for quantitative analysis of diphenylamine, tolylfluanid, propargite and phosalone in liver fractions. Different injector parameters were optimized by an experimental design technique (central composite design). An optimal combination of injector temperature (°C), splitless time (min) and overpressure (kPa) values enabled to maximize the chromatographic responses. Sample preparation was based on protein precipitation using trichloroacetic acid followed by liquid-liquid extraction (LLE) of the pesticides with hexane. All compounds and endrin as internal standard were quantified without interference in selected ion monitoring mode. The calibration curves for diphenylamine, tolylfluanid, propargite and phosalone compounds were linear over the concentration range of 0.1 to 25 μM with determination coefficients (R2) higher than 0.999. A lower limit of quantification of 0.1 μM was obtained for all analytes, i.e. 422.5, 868.0, 876.2 and 919.5 μg/kg of liver fraction (hepatocytes) for diphenylamine, tolylfluanid, propargite and phosalone, respectively. All compounds showed extraction recoveries higher than 93%, with a maximum RSD of 3.4%. Intra- and inter-day accuracies varied from 88.4 to 102.9% and, imprecision varied from 1.1 to 6.7%. Stability tests demonstrated that all pesticides were stable in liver extracts during instrumental analysis (20 °C in the autosampler tray for 72 h) following three successive freeze-thaw cycles and, at −20 °C for up to 12 months. This simple and efficient analytical procedure is thus suitable for metabolism studies or for assessing mammals liver contamination.