Recent advances in analytical techniques have greatly enhanced the depth of coverage, however lipidomic studies are still restricted to analysing only a subset of known lipids. Numerous complementary techniques are used for investigation of cellular lipidomes, including mass spectrometry (MS), nuclear magnetic resonance and vibrational spectroscopy. The development in electrospray ionization (ESI) MS has accelerated lipidomics research in the past two decades and represents one of the most widely used technique. The versatility of ESI-MS systems allows development of methods to detect and quantify a large diversity of lipid species and classes. However, highly targeted and specific approaches can preclude global analysis of many lipid classes. Indeed, experimental procedures are generally optimised for the lipid species, or lipid class of interest. Therefore, careful consideration of experimental procedures is required for characterisation of biological lipidomes. The current review will describe the lipidomic approaches for considering tissue lipid physiology. Discussion of the main sequences in a lipidomics workflow will be presented, including preparation of samples, accurate quantitation of lipid species and statistical modelling.