Fatty acids containing conjugated carbon-carbon double bonds (C═Cs), such as conjugated linoleic acids (CLAs), attract growing research interest due to their bioactivities against diabetes, cancer, and atherosclerosis. Analysis of conjugated fatty acid (CFA) is challenging for existing analytical techniques because it requires determination of geometry (cis ( Z) vs trans ( E)) and location of individual C═C. In this study, we developed a method to achieve confident, fast, and quantitative analysis of CFA isomers from mixtures. This method combines the strength of trapped ion mobility spectrometry (TIMS) for fast isomer separation and the Paternò-Büchi (PB) reaction followed by tandem mass spectrometry (MS/MS) for C═C location determination. Notably, the PB reaction of CFA is regioselective to terminal C═Cs, thus forming diagnostic fragment ions unique to conjugated C═Cs from PB-MS/MS. These fragment ions facilitate identification and quantitation of individual CLA isomers differing in C═C locations, affording limit of identification of 1 nM. Given that PB-MS/MS alone cannot identify the geometry of C═C, TIMS has been employed for characterizing C═C geometry. TIMS is capable to separate various C═C geometric isomers of CLAs, allowing visualization of C═C isomerization during the PB reaction. By coupling the PB-MS/MS with TIMS, two CLA isomers, CLA 18:2(9 Z,11 E) (46.9 ± 1.1%) and CLA 18:2(10 E,12 Z) (53.1 ± 1.1%), are quantified in a commercial CLA supplement.