Rho GTPases including RhoA, Cdc42, and Rac1 are master regulators of cell cytoskeleton dynamic, thus controlling essential cellular processes notably cell polarity, migration and cytokinesis. These GTPases undergo a spatiotemporal regulation primarily controlled by cellular factors inducing both the exchange of GDP for GTP and the hydrolysis of GTP into GDP. Recent findings have unveiled another layer of complexity in the regulation of Rho proteins consisting in their ubiquitylation followed by their proteasomal degradation. Here, we describe how to assess the level of ubiquitylation of Rho proteins in cells, taking Rac1 as an example.