Phosphoinositides are universal regulatory lipid molecules that control multiple signal transduction events and membrane dynamics within the cell. These lipids are present in tiny amounts in biological membranes and are formed and eliminated by multiple inositide kinase and phosphatase enzymes, found in specific cellular compartments. The highly localized roles of these molecules demand the development of methods that can resolve their changes with high temporal and spatial resolution, preferably in living cells. We have been working with protein domains that specifically recognize phosphoinositide isomers and created GFP fusion proteins to visualize lipid changes in intact cells. We also used these domains to interfere with the signalling function of these phospholipids. We will summarize our experience with these methods and review both their benefits and the pitfalls. We will also show new data on imaging of plasma membrane PtdIns4P with the OSH2 tandem PH domain – EGFP fusion protein. We demonstrate the specificity of this probe in vivo within the cells using a type-IV 5-phosphaatase enzyme to convert PtdIns(4,5)P2 to PtdIns4P and simultaneous imaging of PtdIns(4,5)P2 (with PLCd1PH-mRFP) and PtdIns4P (with OSH2-PH-tandem-EGFP). The value of this method to determine which PI 4-kinase enzyme(s) produce the PtdIns4P that is converted to PtdIns(4,5)P2 for agonist-induced Ca2+ signalling will be also shown. Finally, we will review our newest approaches to manipulate Ins(1,4,5)P3 and phosphoinositide levels in specific cellular compartments and will demonstrate how these techniques can help to understand the complexity of phosphoinositide-based signalling processes.