Guanine nucleotide exchange factors (GEFs) of small GTPase (sGTPase) coordinate signal networks in normal cells and dysfunction in cancer. Therefore, effective monitoring of GEF activity is very important for studying the regulation of sGTPase signal transduction. In this study, we developed a 1D 19F NMR-based method for rapid detection of the GEF activity of sGTPases. The activity of Arf6GEF in vitro and cell lysate environment can be conveniently detected by tracking the conformational changes of the Arf6 switch region where a tfmF site-specific 19F labeling at Phe47 was introduced. This strategy could potentially be applied to monitor the conformational change of Arf6 or other sGTPase and detect the activities of sGTPase regulatory proteins in physiology and pathology environments.