In lipidomic research, the accurate quantification and identification of fatty acids in biological samples present methodological and analytical difficulties. From extraction to analysis, the choice of reagents, precision of derivatization, and analytical parameters all influence researchers’ proper understanding of the lipid profile. This is particularly relevant in studies of modern Western diets, where robust analytical methods are critical for assessing human nutritional health. This study aims to optimize key steps in fatty acid extraction, derivatization, and chromatographic analysis. Initial testing was conducted on chicken liver samples, with the intention of applying the optimized method to human samples in subsequent work. By testing different reagent concentrations, we demonstrated that a 46.5% solution of H₂SO₄ in methanol resulted in the most efficient methylation of fatty acids into fatty acid methyl esters (FAMEs), which was measured as higher peak intensities. In contrast, chromatographic separation was primarily influenced by column temperature gradients and injection parameters. The results indicate that a GC oven program starting at 100 °C, with a ramp of 7 °C/min to 240 °C, most effectively separated FAME peaks. Additionally, the use of a phosphate buffer at pH 5.8 during extraction was found to marginally improve peak resolution and amplitude compared to pH 7.0. All results were validated by assessing repeatability through repeated injections, inter-rater reliability, and variation in injection volumes. The novelty of our method lies in its application to soft tissue samples and in the systematic optimization and integration of multiple parameters, such as buffer composition, acid concentration for methylation, and GC/MS settings, which result in a robust, reproducible protocol suitable for high-quality analysis of fatty acids in complex biological matrices.