The (O-acyl)-ω-hydroxy FA (OAHFA) comprise an unusual lipid subclass present in the skin, vernix caseosa, and meibomian gland secretions. Although they are structurally related to the general class of FA esters of hydroxy FA (FAHFA), the ultra-long chain (30-34 carbons) and the putative ω-substitution of the backbone hydroxy FA suggest that OAHFA have unique biochemistry. Complete structural elucidation of OAHFA has been challenging because of their low abundance within complex lipid matrices. Furthermore, because these compounds occur as a mixture of closely related isomers, insufficient spectroscopic data have been obtained to guide structure confirmation by total synthesis.Here, we describe the full molecular structure of ultra-long-chain OAHFA extracted from human meibum by exploiting the gas-phase purification of lipids through multi-stage mass spectrometry and novel, multidimensional ion activation methods. The analysis elucidated sites of unsaturation, the stereochemical configuration of carbon-carbon double bonds, and ester linkage regiochemistry. Such isomer-resolved mass spectrometry guided the first total synthesis of an ultra-long chain OAHFA which, in turn, confirmed the structure of the most abundant OAHFA found in human meibum, OAHFA 50:2. The availability of a synthetic OAHFA opens new territory for future investigations into the unique biophysical and biochemical properties of these lipids.