The seven phosphorylated derivatives of phosphatidylinositol (PtdIns), often collectively referred to as polyphosphoinositides (PPIn), are a minor component of eukaryotic cell membranes. Nevertheless, their synthesis is needed for an ever-increasing spectrum of cellular processes, including regulation of the actin cytoskeleton, chemotaxis, membrane trafficking, glucose uptake, and organelle acidification. PPIn metabolism is regulated dynamically by a network of kinases and phosphatases. Furthermore, synthesis of PPIn can be provoked by external stimuli; for example, the second messenger phosphatidylinositol 3,4,5-trisphosphate rapidly and transiently accumulates in cells challenged with agonists such as PDGF that activate receptor tyrosine kinases. The measurement of PPIn levels in in vivo cultured cells has been vital to our understanding of the metabolism and function of these important signaling molecules; methods are described herein that allow measurement of PPIn levels in culture cells in vivo.