Evaluating total parenteral nutrition (TPN) products for quality assurance and quality control is crucial due to the chemical complexity of its components. With the advent of exploring different approaches for analysing TPN components using tandem mass spectrometry techniques, there is still a need for a robust and reproducible method for industrial routine analyses. This study allows simple, simultaneous determination of 22 fatty acids (FAs) commonly found in TPN components using gas chromatography-mass spectrometry (GC-MS). Five different transesterification techniques were applied for the FA standards and the sodium methoxide in methanol-dimethyl carbonate method was selected due to its good methylation efficiency. Fatty acid methyl esters (FAMEs) were separated in gas chromatography using an HP-5MS UI column with helium as the carrier gas. Mass spectrometry was used to fragment and quantify FAMEs using electron ionization (EI) and selected ion monitoring (SIM) mode. The analytical method was evaluated using the guidelines from the US Food and Drug Agency (FDA) and European Medicines Agency (EMA) in compliance with the International Council for Harmonization (ICH) document Q2(R2). Correlation coefficients (R2) of the calibration curves for FAMEs were 0.99, except for C24:1 n-9 and C24:0, both R2 = 0.98. The limits of detection (LOD) and quantification (LOQ) were found to be 1.69 μg mL-1 and 5.14 μg mL-1, respectively. The linear range was from 3.10-179.9 μg mL-1 for most FAMEs, except for C18:1 n-7 (3.96-224.9 μg mL-1) and C18:1 n-9 (6.30-349.57 μg mL-1). The intra-day and inter-day precision coefficients of variance (CV) of the method were less than 11.10% and 11.30%, respectively. Freeze-thaw cycles and ambient temperature measurements were performed for assessing sample stability. The validated method was applied to analyse major TPN components-fish and olive oils, and an unidentified lipid sample. The presented GC-MS method is simple and robust in the identification and quantification of 22 fatty acids simultaneously in the tested TPN components.