Polysorbates (PS) are widely used as oil-in-water emulsifiers, stabilizers, wetting agents, solubilizers, and dispersants in the agricultural, food, personal care, and pharmaceutical industries due to their cost effectiveness, biocompatibility, formulation flexibility, low toxicity, and good stabilizing and protecting properties. The polysorbates are often pictured as polyoxyethylated sorbitan monoesters of saturated and/or unsaturated fatty acids. In reality, polysorbates are complex mixtures of multiple components, as follows from the reactions involved in their production. In this work, we report a novel application of liquid chromatography-mass spectrometry (LC-MS) for the characterization of polysorbates. This method takes advantage of accurate mass measurements and information on the identity of a fatty acid from "in-source" generated characteristic dioxolanylium ions. The method allowed us to perform quick profiling of fatty acids in PS 20 and 80 which, combined with a computer-aided peak assignment algorithm, facilitated detailed characterization of their constituents. As a major finding, we determined that different samples of PS 20 varied from 0% to 15% in relative amounts of unsaturated oleic acid. Although the consequences of this difference were not fully evaluated in this work, one might expect that PS 20 with larger amounts of oleic acid will be more prone to autoxidation, thus potentially having greater impact on the oxidative degradation of the biotherapeutics it formulates.