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These lipids are distributed across distinct subcellular compartments, where they carry out compartment-specific regulatory functions. Dysregulation of phosphoinositide metabolism is associated with cancer, neurodegenerative diseases, and immune dysfunction. However, their roles remain difficult to investigate owing to technical limitations in lipid detection and manipulation. This review outlines current strategies for modulating, visualizing, and quantifying phosphoinositide pools, including genetic manipulation techniques such as RNA interference, clustered regularly interspaced short palindromic repeats (CRISPR)-based approaches, and optogenetics. It also evaluates visualization tools such as fluorescent biosensors and live-cell imaging techniques, including superresolution microscopy. In parallel, quantitative methods such as thin-layer chromatography and mass spectrometry for profiling phosphoinositide species, including isomer- and acyl-specific variants, are discussed. 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