Lipids comprise large and disparate classes of molecules having very different overall polarities, but the one characteristic they typically have in common is a nonpolar hydrocarbon chain that is hydrophobic in nature. The characteristics of each class of lipid dictate the most appropriate tool and approach for analysis. Since many PLs are inherently charged, the polar head group makes them ideally amenable to soft ionization by electrospray ionization (ESI) coupled to MS. In contrast, triacylglycerols (TAGs) are large neutral molecules that are not easily directly ionized by electrospray ionization mass spectrometry (ESI-MS), and formation of adducts is necessary. Atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) both readily ionize neutral molecules such as TAGs and sterols. This article will outline the characteristics of each class of lipid, and how these affect the choice for the most appropriate tool for their analysis, including special steps and considerations to be taken into account to optimize analysis of each class. Emphasis will be given to neutral lipids (i.e. TAGs), which are the major components of many edible lipid mixtures. The article will also focus on the approaches necessary to derive the desired structural information from lipids. Many of the principles fundamental to lipid analysis in foods and edible oils were developed for analysis of biological samples, but are directly applicable to fats and oils. This article is intended to serve as a guide for decision-making, by discussing the strengths and weaknesses and the benefits and shortcomings of the available options for detailed lipid analysis.