Lipid profiling, which includes fatty acids, phospholipids, glycerides, and cholesterols is extremely important because of the essential role lipids play in the regulation of metabolism in animals. 1H-NMR-based protocols for high-throughput lipid analysis in complex mixtures have been developed and applied to biological systems. Many classes of lipids can be quantitatively analyzed in many sample matrices including serum, cells, and tissues using a simple 1H NMR experiment. In this chapter, we provide protocols for NMR-based lipid profiling including sample preparation, NMR experiments, and quantification using the LipSpin software tool.