The hVps34 phosphatidylinositol (PI) 3-kinase plays an important role in the regulation of vesicular trafficking in the endosomal system. hVps34 associates with a myristylated protein kinase, hVps15. The two proteins are targeting to early endosomal membranes by interactions between hVps15 and activated (GTP-bound) Rab5. This leads to the production of the hVps34 product, PI(3)P, in the endosomal membrane, and subsequent recruitment of FYVE and PX domain-containing effector proteins. This chapter describes the analysis of hVps34/hVps15 interactions with Rab5 in tissue culture cells and in vitro.