Small Ubiquitin-like Modifiers (SUMOs) are an important class of post-translational modification (PTM), modifying target proteins and thereby regulating virtually all nuclear processes. Various proteomics methods exist for profiling SUMO target proteins or SUMOylated lysine residues in a systemic manner, typically based on purification of SUMO followed by mass spectrometry (MS), with most approaches relying on ectopically expressed or mutant SUMO. However, to properly understand the role of SUMO in the context of health and disease, it is necessary to study native and endogenous SUMOylation, rather than relying on genetically engineered systems. Here, we present Native and Endogenous SUMOylation Site Identification using Mass Spectrometry (NESSI-MS), an MS-based proteomics strategy, which entails antibody-based purification of peptides bearing endogenous SUMO2/3 and allows exact mapping of the SUMOylated lysine residues using mass spectrometry.