Heteroatom-centered free radicals are able to transform cis unsaturated fatty acids to the thermodynamically more stable, but unnatural, trans configuration. The "geometrical" radical stress can be estimated in biological samples using trans fatty acid isomers as lipid markers. Regioselectivity is an important feature of the "geometrical" radical stress, because the supramolecular organization of the polyunsaturated fatty acid moieties of phospholipids can lead to preferential monotrans isomer formation. The regioisomer recognition is a crucial step, which is helped by appropriate molecular libraries available through radical-based synthetic methodologies. Cholesteryl linoleate and arachidonate esters are interesting targets for their biochemical connection with membrane phospholipid turnover and their well-known roles in cardiovascular health. The synthesis of monotrans isomers of PUPA cholesteryl esters was achieved by a thiyl radical-catalyzed cis-trans isomerization. Valuable NMR, IR, and Raman spectroscopic data have been collected for promising application in metabolomics and lipidomics. The identification of monotrans cholesteryl ester isomers was carried out in human plasma by GC, Raman, and IR analyses, demonstrating for the first time the presence of specific regiosiomers connected to free radical stress. This work helps to highlight the chemical biology approach for the access to molecular libraries applicable to biomarker development, and the cis-trans isomerization as a relevant process to be integrated in the puzzling scenario of free radical-mediated lipid modifications.