Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is a phospholipid enriched on the cytoplasmic leaflet of the plasma membrane, where it plays important roles in membrane trafficking and cytoskeletal dynamics through proteins that directly bind to it. PI(4,5)P2 can be metabolized to other phosphorylated forms of phosphatidylinositol to regulate numerous processes such as cell growth and development. PI(4,5)P2 can also be hydrolyzed to generate the second messengers diacylglycerol (DAG) and inositol triphosphate (IP3). Altered metabolism or mislocalization of PI(4,5)P2 can perturb one or more of its functions and contribute to disease states. Here, we present a protocol to visualize and quantify the localization of PI(4,5)P2 in live cells. The protocol uses a highly specific PI(4,5)P2 protein binding domain coupled to enhanced green fluorescence protein (PH-PLCD1-GFP), enabling localization and quantification of cytosol-facing PI(4,5)P2 to be determined. Localization and quantification of the PH-PLCD1-GFP, PI(4,5)P2 specific probe, is enabled by fluorescence imaging and confocal microscopy. This approach can be used to study the dynamics of PI(4,5)P2 localization temporally in live cells under both physiological and pathological conditions. Key features • Protocol for the quantification of PI(4,5)P2 membrane localization in live cells. • Uses the expression of the highly specific PH-PLCD1-GFP, PI(4,5)P2 probe, in cells, followed by fluorescence image acquisition using confocal microscopy and subsequent image processing. • Adaptable to various cell types and experimental conditions. • Presents detailed instructions for reagent preparation, fluorescence measurement, and quantification.