<p>Among stable isotope measurement strategies at natural abundance, position-specific isotope analysis (PSIA) is a valuable approach for food authentication and metabolism understanding. In this context, carbon-13 nuclear magnetic resonance (irm-¹³C NMR) spectroscopy has emerged as an efficient tool. It enables the determination of the absolute ¹³C intramolecular composition (δ¹³C_i) when all NMR signals are well-resolved and the global ¹³C content (δ¹³C_g) is known. However, in the case of fatty acids with eight carbons or more, ¹³C-NMR can only resolve six carbon signals. δ¹³C_i values are thus not attainable without specialized methods. To overcome this limitation, we developed a novel methodology that leverages the concept of isotopic intramolecular referencing by using the methoxy group in the fatty acid methyl esters (FAMEs) as a reference. We thus determined δ¹³C_i values with improved trueness and repeatability without requiring δ¹³C_g measurement. Combining this enhancement with the acquisition of ¹³C NMR spectra using an adiabatic INEPT pulse sequence offered a more time-efficient, sample-efficient, and sustainable approach, rendering the entire process greener. We successfully applied this approach to FAMEs from butter and coconut oil. Subsequent studies will evaluate the effectiveness of the novel isotopomic biomarkers derived from our method in authenticating foodstuffs and elucidating metabolic differences among fatty acids. Additionally, the same intramolecular isotope referencing approach will be of interest for studying other molecules and isotopes.</p> Graphical Abstract <p></p>