Ubiquitination of proteins has emerged as a vital posttranslational modification at the crux of numerous signalling pathways, regulating them in various ways. Most members of the small GTPase family including Ras and Rho proteins are regulated by GEFs, GAPs, and RhoGDIs that modulate their cycling between the active and inactive states. Ubiquitination has added another layer to the regulation of small GTPases. Recently, we have uncovered that inhibitors of apoptosis (IAPs) function as direct E3 ubiquitin ligases for Rho GTPase Rac1 and target it for proteasomal degradation. Here, we describe in vitro and in vivo ubiquitination assays for detecting the conjugation of ubiquitin to Rac1 by XIAP and cIAP1.