Glycerophospholipids are the structural building blocks of the cellular membrane. In addition to creating a protective barrier around the cell, lipids are precursors of intracellular signaling molecules that modulate membrane trafficking and are involved in transmembrane signal transduction. Phospholipids are also increasingly recognized as important participants in the regulation and control of cellular function and disease. Analysis and characterization of lipid species by mass spectrometry (MS) have evolved and advanced with improvements in instrumentation and technology. Key advances, including the development of "soft" ionization techniques for MS such as electrospray ionization (ESI), matrix-assisted laser desorption/ionization (MALDI), and tandem mass spectrometry (MS/MS), have facilitated the analysis of complex lipid mixtures by overcoming the earlier limitations. ESI-MS has become the technique of choice for the analysis of multi-component mixtures of lipids from biological samples due to its exceptional sensitivity and capacity for high throughput. This chapter covers qualitative and quantitative MS methods used for the elucidation of glycerophospholipid identity and quantity in cell or tissue extracts. Sections are included on the extraction, MS analysis, and data analysis of glycerophospholipids and polyphosphoinositides.