Signaling enzymes are typically regulated by a variety of post-translational modifications. One such enzyme, protein tyrosine phosphatase (PTP) 1B, which plays an important role in metabolic and growth signaling, has recently been shown to be inhibited by posttranslational modifications by ubiquitin-related proteins of the SUMO family. We have adapted several approaches to analyzing the sites and effects of sumoylation of PTP1B in cells and in vitro. The principal outline of the assays should be applicable to other enzymes as well.