Determining the unique isomeric structures of lipids is vital to understanding their biochemical functions in organisms. Distinct lipid isomers have been directly linked to a variety of disease states and properties of cellular membranes. Being able to distinguish between C=C positional isomers has proven to be particularly difficult. Traditional methods of C=C position elucidation included complex derivatization of the parent lipids before analysis could be performed. However, many new methodologies have become popular in the past 10 years for more simple, high-throughput, reproducible analyses of lipid fatty acids. Tandem mass spectrometry methods such as ozone-induced dissociation (OzID) and the Paternò−Büchi (PB) reaction have made significant contributions to the workflow of lipid isomer analysis. Both methods have evolved substantially from their initial proposals, and have produced creative solutions to many problems that have arisen. These identification and quantification methods have resulted in greater understandings of biological lipidomes and have aided in the comprehension of medical evaluations.