Oxysterols are oxygenated forms of cholesterol. This definition can, however, be expanded to include oxygenated derivatives of plant sterols and also of cholesterol precursors. Oxysterols are formed in the first steps of cholesterol metabolism and also from cholesterol by reactive oxygen species. Oxysterols were once thought of as simple intermediates, or side-products, in the conversion of cholesterol to hormonal steroids and bile acids, however, they have subsequently been shown to be biologically active molecules in their own right. In this article we will discuss methods of oxysterol analysis including “classical” gas chromatography–mass spectrometry (GC–MS) methods and more recent liquid chromatography (LC)–MS methods. Our main focus, however, will be on analytical methods based on “charge-tagging” and LC–tandem mass spectrometry (MS/MS or MSⁿ) which we have developed over the last decade in our laboratory. Examples will be given of oxysterol analysis in brain, cerebrospinal fluid (CSF) and blood. The advantages and disadvantages of the various methods of oxysterol analysis will be discussed. This article is part of a Special Issue entitled Lipodomics and Imaging Mass Spectrometry.