Rationale: Eicosanoids are short-lived bio-responsive lipids produced locally from oxidation of polyunsaturated fatty acids (FAs) via a cascade of enzymatic or free radical reactions. Alterations in the composition and concentration of eicosanoids are indicative of inflammation responses and there is strong interest in developing analytical methods for the sensitive and selective detection of these lipids in biological mixtures. Most eicosanoids are hydroxy FAs (HFAs), which present a particular analytical challenge due to the presence of regioisomers arising from differing locations of hydroxylation and unsaturation within their structures. Methods: In this study, the recently developed derivatization reagent 1-(3-(aminomethyl)-4-iodophenyl)pyridin-1-ium (4-I-AMPP+) was applied to a representative set of HFAs including bioactive eicosanoids. Photodissociation (PD) mass spectra obtained at 266 nm of 4-I-AMPP+-modified HFAs exhibit abundant product ions arising from photolysis of the aryl–iodide bond within the derivative with subsequent migration of the radical to the hydroxyl group promoting fragmentation of the FA chain and facilitating structural assignment. Results: Representative polyunsaturated HFAs (from the hydroxyeicosatetraenoic acid and hydroxyeicosapentaenoic acid families) were derivatized with 4-I-AMPP+ and subjected to a reversed-phase liquid chromatography workflow that afforded chromatographic resolution of isomers in conjunction with structurally diagnostic PD mass spectra. Conclusions: PD of these complex HFAs was found to be sensitive to the locations of hydroxyl groups and carbon–carbon double bonds, which are structural properties strongly associated with the biosynthetic origins of these lipid mediators.