The present research is focussed on the optimization of a fast and economically convenient method for the sample preparation and gas chromatographic separation of fatty acids contained in lipidic samples. This, as part of a wider project that has, as ultimate goal, the automation of this specific analytical procedure. The developed approach was applied to the analysis of cod liver oil, a highly complex lipid characterized by a wide nutritional interest. Derivatization was carried out by using low quantities (μL amounts) of a reagent and solvent, while the derived fatty acid methyl esters (FAMEs) were separated in 120 s on a 10 m × 0.1 mm i.d. polar micro-bore column. The total analysis time required for six samples was approximately 45 min (7.5 min/sample), considering a simultaneous process of methylation and GC separation of previously prepared samples. The results obtained were compared to those derived from conventional applications on the same sample. With regard to the validation of the rapid method, peak area/retention time repeatability, linear range, limit of detection (LOD) and quantitation (LOQ) were determined. Peak assignment was carried out by exploiting bidimensional group-type mapping information obtained in a comprehensive gas chromatographic application.