The GPCR-coupled sphingosine-1-phosphate (S1P) receptors regulate a number of important cell �functions, including proliferation, migration, and adhesion. Since these processes require dynamic regulation of the actin cytoskeleton, the ability to monitor S1P-dependent activation of the Rho family GTPases is critical for our understanding of S1P signaling. Herein, we provide methods for the GST pull-down-based assay used to measure Rho, Rac, and Cdc42 activity in cultured cells treated with S1P.