Small GTPases of the Rho family are important modulators of the cytoskeleton and regulate morphogenetic cell movements during embryonic development. In the Xenopus embryo, Rho signaling contributes to the regulation of convergent extension (CE) movements in gastrula and neurula stages as well as to tissue separation (TS). Here we describe a method that allows the detection of activated (GTP-bound) Rho in fixed Xenopus tissue. The assay makes use of a fusion protein of Rhotekin and Green-Fluorescent-Protein (RBD-GFP), which is produced in bacteria and can be purified biochemically. This technique allows a temporal and spatial analysis of Rho signaling in the developing embryo. Developmental Dynamics 238:1407–1411, 2009. © 2009 Wiley-Liss, Inc.