Shotgun lipidomics, based on multi-dimensional mass spectrometric array analyses after multiplexed sample preparation and intrasource separation, has been recently advanced to a mature technique for the rapid and reproducible global analysis of cellular lipids. At its current stage, this technology enables us to analyze more than 20 lipid classes and thousands of individual lipid molecular species directly from lipid extracts of biologic samples. Following a brief introduction to the foundations underlying this rapidly expanding technology, we present detailed protocols used for the identification and quantitation of plasma triglycerides, determination of the human heart lipidome, and analysis of cellular cardiolipin molecular species. Through the use and practice of shotgun lipidomics, new insights into the cardiovascular pathobiology manifest in diabetes and the metabolic syndrome can be accrued.