Inhibitor of apoptosis (IAP) proteins are a family of evolutionarily conserved proteins that regulate apoptosis as well as other cellular processes. The functions of many IAPs are defined by their RING domains, which possess E3 ubiquitin ligase activity and promote proteasomal degradation of an increasing number of target proteins. In this chapter, we describe the methods used in our laboratories to study the IAP's E3 activity.