Objective: To establish a GST pulldown assay for detection of Daam1 activity in eukaryotic cells. Methods: Prokaryotic expression vector containing fusion protein GST-RhoA was constructed. The correct plasmid was transfected into E. coli. BL21 stain and inducted the expression of the fusion protein by IPTG. SDS-PAGE and Western blotting were utilized to determine the corresponding recombinant proteins. The fusion protein with glutathione beads was pulled down to detect the activity of Daam1 in HEK293T cells. Results: GST-RhoA vector was successfully conducted and stably expressed fusion protein GST-RhoA. The activity of Daam1 was detected by GST pulldown assay,showing high activity of Daam1 in HEK293T cells. Conclusion: The activity of Daam1 was successfully detected by GST pulldown assay. This experimental scheme can be used for further studies of active Daam1 targeting in eukaryotic cells.