Wnt5a is a representative ligand that activates the β-catenin-independent pathway of Wnt signaling in mammals. This pathway might be related to planar cell polarity signaling inDrosophila. Because reliable biochemical assays to measure Wnt5a pathway activity have not yet been established, we examined whether Wnt5a signaling stimulates focal adhesion turnover in migrating cells using live immunofluorescence imaging and immunocytochemical analysis. These assays demonstrated that the Wnt5a pathway cooperates with integrin signaling to regulate cell migration and adhesion through focal adhesion dynamics.