Phosphorylated derivatives of phosphatidylinositol (PtdIns), known as phosphoinositides (PIs), are essential regulators of nuclear functions, cytoskeletal dynamics, cell signaling and membrane trafficking. These lipids are found on the cytosolic face of intracellular membranes but can also be detected in membrane-free regions of the nucleoplasm. Their downstream effectors include several proteins that contain various PI-specific domains. Because impaired PI metabolism is associated with disorders such as cancer, cardiovascular disease and immune dysfunction, there is currently great interest in studying PIs and their metabolic enzymes. Here we describe strategies and techniques for quantitative and qualitative measurement of PIs, for characterization of specific PI-binding proteins and for determination of PI kinase and phosphatase activities in vitro and in vivo.