Polysorbate (PS) is a widely used polymeric excipient in biotherapeutic formulations to stabilize and protect protein drugs. Commercial PS is a highly heterogeneous mixture of structurally related components. PS composition can impact the stabilizer performance of PS in formulated protein drugs. Characterization of PS heterogeneity is, however, analytically challenging. In this work, a high-throughput screening protocol is presented for the profiling of the PS-80 polysorbate form using high resolution mass spectrometry (HRMS) coupled with a rapid hydrogen/deuterium (H/D) exchange in deuterated methanol. The protocol takes advantage of accurate mass measurements from HRMS analysis and utilizes H/D exchange-induced mass shifts that are characteristic to structures (particularly the number of terminal hydroxyl groups) of PS molecules to definitively identify species. In particular, mass shifts caused by deuterium uptake were used (1) to confirm molecular identities assigned by accurate mass measurements (which adds an extra level of identification confidence) and (2) to differentiate isomers that have an identical mass (thus, undistinguishable by high mass accuracy), but differ in the number of terminal hydroxyls. These data were input to an automated searching algorithm against a molecular mass database covering over 17000 potential PS-80 molecular species. The identified species were then visualized with Kendrick Mass Defect plots. The analysis protocol identified and profiled over 180 species from PS-80 samples in a high-throughput fashion without requiring chromatographic separation to reduce complexity of mixtures or tandem mass spectrometric analysis to conduct structural elucidation.