To identify Vap33 binding proteins, we purified Vap33-containing protein complexes from Drosophila embryos, using the single-step GFP-trap bead purification from a fly line expressing YFP-tagged Vap33, followed by nanoLC-MS/MS analysis of the interactors. This study revealed the binding of Vap33 to RtGEF and Git, which we further connected to the regulation of Hippo signaling.