This Application Note describes the analysis of complex lipids in blood plasma. Lipids are extracted using a bi-phasic solvent system of methanol, methyl tert‑butyl ether (MTBE), and water followed by separation and data acquisition of isolated lipids using reversed-phase liquid chromatography coupled to quadrupole time‑of‑flight mass spectrometry (RPLC/Q-TOF MS). This workflow permits detection of over 270 unique lipid species covering the main plasma lipid classes.