Objective:To establish a GST pulldown assay that can detect the activated Rab35 in eukaryotic cells. Methods:We constructed prokaryotic expression vector containing fusion protein GST-RUN. The correct plasmid was transfected into Ecoli. BL21 stain and the expression of the fusion protein was inducted by IPTG. SDS-PAGE and Western blotting were utilized for determination of the corresponding recombinant proteins. Then we pulled down the fusion protein with glutathione beads to detect the activated Rab35 in HEK293T cells. Results:The prokaryotic expression vector GST-RUN was successfully constructed and expressed fusion protein GST-RUN stably. GST pulldown assay showed high activated Rab35 in HEK293T cells. Conclusion:The activated Rab35 was successfully detected by GST pulldown assay. This experimental scheme can be used for further study of active Rab35 targeting in eukaryotic cells.