Endocannabinoids and related N-acyl ethanolamine-derived lipids affect a diverse array of physiological processes and pathological conditions. In the roundworm C. elegans, several endocannabinoid-like molecules have been implicated in regulating axon regeneration, energy balance and food intake as well as aging. One such molecule oleoylethanolamine (OEA) has been shown to promote life extension through nuclear receptor signal transduction, and its accurate quantitation therefore is of high interest. Using a combination of electron impact ionization (EI) and collision induced dissociation coupled to gas chromatography (GC), we found unique fragmentation ions of OEA and designed a specific MRM method for its accurate quantification. Our method should provide a reproducible and robust way to measure OEA dynamics under different genetic, pharmacologic and environmental perturbations.