Rho GTPases play critical roles in regulating podocyte cytoskeleton and their alteration leads to foot process effacement. However, their signaling networks remain poorly understood. To gain better insights into Rho GTPases signaling in human podocyte, we implemented the high-throughput proximity-dependent biotinylation assay (BioID) using 9 baits of Rac1, Cdc42 and RhoA covering their various nucleotide loading states: Control wild-type Rho GTPases (RhoA WT, Rac1 WT, and Cdc42 WT), nucleotide-free (NF) mutants (RhoAG17A, Rac1G15A, and Cdc42G15A), constitutively active mutants (RhoAG14V, Rac1G12V, and Cdc42G12V).