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Rationale: Various medium formulations contain essential fatty acids at concentrations ranging from 10 to 100 mg/L. Accurate and precise lipid measurement in media is crucial for monitoring media quality and conducting studies on lipids in the context of cell culture. This study employed two‐dimensional gas chromatography (GC × GC) analyses to offer enhanced resolution, sensitivity, and separation performance compared to GC. Methods: Quantification of fatty acid methyl esters (FAMEs) in a medium was conducted using GC × GC combined with a high‐resolution mass spectrometer and flame ionization detector, considering potential interference from nonionic surfactant Tween 80, which was precipitated and removed by optimizing the concentration of cobalt thiocyanate (CTA) solution during pretreatment. This advanced analytical approach enabled identification of cis and trans isomers of identical molecular weights and determination of the location and number of double bonds in the same carbon number structure. Results: Our analysis identified 36 FAMEs within the C6–C24 region, and a 5% CTA solution was optimal for efficient removal of Tween 80 during lipid extraction. Additionally, this advanced method minimized FAME contamination and loss during pretreatment, thereby significantly reducing the sample volume required to detect trace levels of FAMEs. This improvement led to a fatty acid recovery rate of 106% while maintaining the average relative standard deviation for the target FAMEs of about 3%. Conclusions: Our research paves the way for future investigation into medium quality control and the role of fatty acids in cell culture. This offers the possibility for economical and effective trace quantification of fatty acids in complex media. [ABSTRACT FROM AUTHOR]