Class I phosphoinositide-3-kinase (PI3K) isoforms generate the intracellular signaling lipid, phosphatidylinositol(3,4,5)trisphosphate (PtdIns(3,4,5)P₃). PtdIns(3,4,5)P₃ regulates major aspects of cellular behavior, and the use of both genetic and pharmacological intervention has revealed important isoform-specific roles for PI3Ks in health and disease. Despite this interest, current methods for measuring PtdIns(3,4,5)P₃ have major limitations, including insensitivity, reliance on radiolabeling, low throughput and an inability to resolve different fatty-acyl species. We introduce a methodology based on phosphate methylation coupled to high-performance liquid chromatography–mass spectrometry (HPLC-MS) to solve many of these problems and describe an integrated approach to quantify PtdIns(3,4,5)P₃ and related phosphoinositides (regio-isomers of PtdInsP and PtdInsP₂ are not resolved). This methodology can be used to quantify multiple fatty-acyl species of PtdIns(3,4,5)P₃ in unstimulated mouse and human cells (≥10⁵) or tissues (≥0.1 mg) and their increase upon appropriate stimulation.