Binary stable isotope labeling couple with LC-ESI-MS has been used as a powerful non-targeted approach for the relative quantification of lipids, amino acids, and many other important metabolite classes. A multiplexed approach using three or more isotopic labeling reagents greatly reduces analytical run-time while maintaining excellent sensitivity and reproducibility. Three isotopic cholamine labeling reagents have been developed to take advantage of the pre-ionized character of cholamine, for ESI, and the ease by which stable isotopes can be incorporated into the cholamine structure. These three cholamine labeling reagents have been used to relatively quantify three fatty acid samples simultaneously. The quantification resulted in the observation of 12 fatty acids that had an average absolute error of 0.9% and an average coefficient of variation of 6.1%. Caged versus cage-free isotope labeling experiments showed that cage-free eggs have an increased level of omega-3 fatty acids as compared to caged eggs. This multiplexed fatty acid analysis provides an inexpensive and expedited tool for broad-based lipid profiling that will further aid discoveries in the mechanisms of fatty acid action in cells.