Polysorbate 80 (PS80), a chemical substance composed of sorbitol, ethylene glycol, and fatty acids, is commonly used in pharmaceutical drug products to stabilize formulations. However, recent studies have demonstrated that PS80 may hydrolyze over time and the released free fatty acids (FFAs) may lead to particle formation. Naming conventions of fatty acids in current pharmacopeia and in products’ certificates of analysis (CoA) of PS80 do not typically distinguish between isomeric species of fatty acids in PS80. Thus, methods to fully characterize the fatty acid species present in PS80 raw materials are needed to enhance quality control strategies of pharmaceuticals using PS80. Here, extended effort is taken to characterize fatty acids in hydrolyzed PS80 raw materials and elucidate the identities of isomeric fatty acid species. In this work, a method was developed and optimized for separation and detection of fatty acids in alkaline hydrolyzed PS80 raw materials using ultra performance liquid chromatography (UPLC) with ultra-violet (UV) detection and evaporative light scattering detection (ELSD). Fatty acids not specified in the current pharmacopeias were detected in PS80 raw material by the developed LC-UV-ELSD method including conjugated forms of linoleic and linolenic fatty acid species. Their identities were orthogonally confirmed by retention time agreement with analytical standards, accurate mass by high resolution mass spectrometry, UV absorbance, and proton nuclear magnetic resonance spectroscopy. The detected conjugated fatty acids are theoretically more hydrophobic and less soluble than their unconjugated counterparts and may increase the propensity of PS80 to form particles upon hydrolysis. This work highlights the need for better quality control of PS80 raw material, as it may eventually play a critical role in product quality of therapeutic proteins.