Fatty acids are a structurally diverse category of lipids with a myriad of biochemical functions, which includes their role as building blocks of more complex lipids (e.g., glycerophospholipids and triacylglycerols). Increasingly, the analysis of fatty acids is undertaken using liquid chromatography-mass spectrometry (LC-MS), due to its versatility in the detection of lipids across a wide range of concentrations and diversity of molecular structures and masses. Previous work has shown that fixed-charge pyridinium derivatives are effective in enhancing the detection of fatty acids in LC-MS workflows. Herein, we describe the development of two novel pyridinium fixed-charged derivatization reagents that incorporate a photolabile aryl iodide that is selectively activated by laser irradiation inside the mass spectrometer. Photodissociation mass spectra of fatty acids conjugated to 1-(3-(aminomethyl)-4-iodophenyl)pyridin-1-ium (4-I-AMPP+) and 1-(4-(aminomethyl)-3-iodophenyl)pyridin-1-ium (3-I-AMPP+) derivatives reveal structurally diagnostic product ions. These spectra feature radical-directed dissociation of the carbon-carbon bonds within the fatty acyl chain, enabling structural assignments of fatty acids and discrimination of isomers that differ in site(s) of unsaturation, methyl branching or cyclopropanation. These derivatives are shown to be suitable for hyphenated LC-MS methods, and their predictable photodissociation behavior allows de novo identification of unusual fatty acids within a biological context.