Fatty acids (FA) serve many important roles in human health, acting as energy reserves, signaling molecules, membrane components, and more. FA are also associated with disease risk, with individual FA associated with increased risk of cardiovascular disease or shown to influence inflammatory diseases and type 2 diabetes. In order to better understand the role of individual FA in human health and disease, it is important to quantitatively assess these FA in biological samples. However, biological samples contain a large variety of FA, including many positional and geometric isomers, with over 65 different FA including trans-FA (TFA) being reported in humans. The large number of cis- and trans-isomers pose analytical challenges, because these isomers have the same molecular mass and very similar chemical structures. Herein we provide an overview of technologies used to assess fatty acid positional and geometric isomers. We combine the CDC isotope dilution-gas chromatography-negative chemical ionization-mass spectrometry (ID-GC-NCI-MS) method with Ag+ ion HPLC to separate and identify over 50 geometric FA isomers in human plasma. We also utilize mCPBA to epoxidize FA double-bonds and analyze the FA-epoxides via GC-MS/MS using multiple reaction monitoring to assess positional FA isomers in human plasma. Fragmenting the epoxides allows us to distinguish C16:1n-7t and C16:1n7c from 10 other C16:1 isomers present when using selected ion monitoring. These approaches allow us to assess both geometric and positional isomers in order to identify unknown FA of interest in blood.