Sav1 scaffold protein regulates Hippo signaling pathway, which plays an important role in the organ size control and the tumorigenesis. Sav1 interacts with Mst1/2 and Lats1, which comprises a core component of Hippo pathway. In this study, we tried to identify weak interacting proteins by proximity-labeling techniques. Promiscuous biotin ligase, called BioID2, biotinylates the proteins within about 10 nm. BioID2 gene was ligated to 5' end of Sav1 cDNA, which was inserted into tetracycline-inducible expression vector. This expression vector was stably introduced into HEK293 cells. Tetracycline-induced expression was confirmed by western blotting and cell immunostaining. This BioID2-Sav1 was confirmed to interact with Mst1 by co-immunoprecipitation assay. Next, biotinylated proteins was pulled down using biotin binding partner, strep-tactin, conjugated beads. The pulled down fraction contained at least BioID2-Sav1 and endogenous Sav1 (Sav1 homodimerizes). As the results of western blotting by strep-tactin-HRP and silver staining, more than 8 proteins were biotinylated and pulled down. This indicates that the BioID2 method may be useful for identification of novel Sav1 interacting proteins.