To identify Lgl binding proteins, we purified Lgl-containing protein complexes from cultured Drosophila S2 cells, using the single-step streptavidin purification from stable cell lines expressing SBP-tagged Lgl, followed by nanoLC-MS/MS analysis of the interactors. This study revealed the binding of Lgl to Vap33, which we further connected to the function of the vacuolar ATPase and regulation of Notch signaling.