Small ubiquitin-related modifier (SUMO) is an ubiquitin-like protein that is covalently attached to a variety of target proteins. Unlike ubiquitination, sumoylation does not target proteins for proteolytic breakdown, but is instead involved in regulating a variety of different protein functional properties, including protein-protein interactions and subcellular targeting, to name a few. Protein sumoylation has been particularly well characterized as a regulator of many nuclear processes as well as of nuclear structure, making the characterization of this modification vital for understanding nuclear structure and function. Because sumoylation plays an important role in regulating so many important cellular processes, there has been intense interest in identifying new proteins that are targets of this modification and determining what role sumoylation plays in regulating the protein functions. This chapter presents methodologies for determining whether a particular protein is a substrate of sumoylation, and for identifying the lysine residue(s) where the modification occurs.