Lipidomics can be regarded as the quantitative profiling of the entire lipid composition of a defined system, be that a cell, tissue, biofluid or intact organism. Lipidomics is a descendent of what was previously known as “metabolite profiling” and is a relative of the related “omic” disciplines of genomics, transcriptomics and proteomics. Lipidomics can be regarded as a sub-division of metabolomics. In this chapter we will discuss the current methodologies popular in lipidomics, highlighting gas-chromatography mass spectrometry (GC-MS), liquid-chromatography MS (LC-MS) and direct-infusion, also known as “shotgun” MS (DI-MS). We will also introduce newer methods including low-flow-rate-LC MS, ion mobility MS (IM-MS) and MS imaging (MSI), described in detail in later chapters. Chemical derivatisation is not a new idea to lipidomics; however, its use goes in and out of vogue, hence we also introduce some of its benefits and disadvantages here and in a later chapter.