Lipidomic methodologies, in conjunction with biochemical isolation of intracellular organelles such as the nucleus, have identified a wide variety of nuclear lipid species. The nuclear lipid composition, for example, exhibits daily changes and is controlled both by the circadian clock and feeding behavior. However, the functions of many of these lipids in the nucleus remain elusive. In this chapter we describe in detail the procedure for nuclear lipidomics; from biochemical isolation of nuclei from mouse liver and cultured cells to the unbiased lipid identification and quantification using Multi-Dimensional Mass Spectrometry Shotgun Lipidomics (MDMS-SL). The approach described herein is applicable with some modifications for different tissues and various animal models, as well as a wide variety of cultured cells. It is expected to yield new insights into various aspects of nuclear lipid composition and function in the future.