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Many and various experimental techniques have been developed to fully analyze the intracellular signaling pathways of membrane phosphoinositides and their water‐soluble derivatives. This chapter concentrates mainly on the range of lipid‐derived, water‐soluble signaling molecules that can be produced in cells from these membrane phosphoinositides, for which we and others have proposed biological roles. These include lysophosphatidylinositol, produced via phospholipase A 1/2 activities on phosphatidylinositol; cyclic inositol phosphates, produced via phosphatidylinositol/lysophosphatidylinositol‐specific phospholipase C activities; and glycerophosphoinositols, produced via lysophospholipase A 2/1 activities on their corresponding lysophosphoinositides. While the methodologies described in this chapter are aimed more specifically at the separation, identification, and quantification of monophosphorylated glyceropho sphoinositols and other similarly charged inositol‐containing products of the membrane phosphoinositides in cell extracts, they can be equally well applied tothe full range of lysophosphoinositides, glycerophosphoinositols, inositol phosphates, and further inositol‐containing water‐soluble products of the phosphoinositides (e.g., cyclic inositol phosphates, methylphosphoinositol phosphates).