VPS34 is the only class III phosphatidylinositol-3-kinase (PI3K) in mammalian cells and produces the vast majority of cellular phosphatidylinositol-3-phosphate [PI(3)P]. PI(3)P is a key signalling lipid that plays many membrane trafficking roles in processes such as endocytosis and autophagy. VPS34 is a key cellular regulator, loss of function can have catastrophic effects and is embryonic lethal (Zhou et al., 2011). The levels of cellular PI(3)P can be determined by fluorescent staining techniques and can be used to monitor effects upon VPS34 activity, however it is important to verify that any changes are mediated by VPS34, particularly as alternate pathways of PI(3)P production are possible such as via class II PI3Ks (Devereaux et al., 2013). Assaying VPS34 activity directly in vitro can be a key stage in delineating the action of a particular stimulus.