Abstract: Naturally occurring lipid double bond isomers carry the signature of lipid dietary history, biosynthesis, and catabolism. The mass levels of these isomers directly influence membrane and cellular functions, thereby having significant impacts on the development of pathologies such as cancer, cardiovascular disease and type 2 diabetes. However, identification of the positions of carbon-carbon double bonds within lipids and quantification of these identified isomeric species are always challenging. The current analytical approaches to elucidation of lipid double bond isomers require chemical derivatization, chromatographic separation, specialized instrumentation (high-energy collision induced dissociation (CID), multistage mass spectrometry, ozone electrospray ionization, etc.), or interpretation of complex fragmentation patterns^1-4. In the current study, we present a mechanically straightforward and operationally simple approach to determine the double bond positions in unsaturated fatty acids and potentially in their more complex derivatives. Natural loss of carbon dioxide or water from carboxylic acid (i.e., decarboxylation or dehydration, respectively), rarely occurs from simple carboxylic acids due to the requirement of high activation energy. Decarboxylation or dehydration from deprotonated fatty acids, however, is not unusual upon CID in negative ion mass spectrometry even at low collision energy. Intriguingly, by using multi-dimensional mass spectrometry based shotgun lipidomics with collision energy as the desired dimension^5-6, we found that the extent to which the deprotonated fatty acids underwent decarboxylation and/or dehydration upon CID was dependent on the degree of unsaturation and in particular the positions of the double bonds. These findings in combination with the known knowledge of double bond distribution in naturally occurring unsaturated fatty acids allow us to identify the double bond locations. The mechanism underlying the double bond position-dependent decarboxylation or dehydration was investigated in the study. The standard curve for each suite of isomeric unsaturated fatty acid pairs could be established by fragmenting individuals and their mixtures at a broad range of ratios upon collision induced dissociation under differential low collision energies by using an electrospray ionization mass spectrometer. Sequential scans and data processing for standard curve establishment were performed as previously described⁶. The standard curves established afford not only the identification of the double bond position in unsaturated fatty acids but also the quantitation of the composition of the coexisting isomeric pairs, which is inaccessible or difficult to perform by most of the up-to-date approaches available. Collectively, we present a novel method that offers the opportunity for quick and direct determination of double bond position and isomeric composition of unsaturated fatty acids by electrospray ionization tandem mass spectrometry without derivatization and specialized instrumentation. This method can be potentially applied to the determination of double bond positions in unsaturated complex lipids such as in fatty acyl constitutes in phospholipids, which, along with the readily available assignment of fatty acyl constitute regiospecificity, permits a near complete structural characterization of complex lipids.