To study composition and heterogeneity of insoluble subvisible particles in Mab formulations resulting from degradation of polysorbate 20 and to develop a better understanding of the mechanisms of polysorbate degradation leading to particle formation. In this study, we exploit the potential of Raman microscopy for chemical identification of particles in monoclonal antibody formulations. Through a combination of experiments and density functional theory (DFT) calculations, we identified unique spectral marker bands for insoluble degradation products of polysorbate 20. We first applied our methodology to identify particles in model systems containing complex mixtures of fatty acids and then to subvisible particles in antibody formulations stored at 5°C for several years. Most of the subvisible particles identified were comprised of mixtures of fatty acids with no observable signal from fatty acid esters consistent with hydrolysis being the predominant degradation mechanism leading to particulate formation under these storage conditions. Our methodology is generally applicable for identification of particles in antibody formulations and, in particular, has the potential to give detailed information about particle heterogeneity and insight into mechanistic aspects of particle formation.