Fatty Acid Esters of Hydroxy Fatty Acids (FAHFAs) are a recently discovered class of biological active lipids with anti-diabetic and anti-inflammatory functions. Given that structure and function are intimately related, we report here the use of direct infusion multi-stage hybrid tandem mass spectrometry involving sequential Collisional Activated Dissociation (CAD) and 213 nm UltraViolet PhotoDissociation (UVPD), as a novel technique for the unambiguous denovo identification and detailed structural characterisation of FAHFA lipid ions, including determination of the esterified fatty acid identity, the hydroxy fatty acid identity and position of esterification, and localization of the site(s) of endogenous unsaturations, without need for chromatographic separation or authentic reference standards. The utility of this approach is demonstrated for the identification of individual FAHFA lipids introduced to the mass spectrometer in positive ionization mode as their lithiated adducts, as well as from mixtures containing isomeric FAHFA species with differing esterification sites, including those that are not resolved by current liquid chromatography methods.