In man the brain represents about 2% of the body weight, but contains 25% of the body's cholesterol. Cholesterol itself does not cross the blood-brain barrier and is synthesised in situ. Excess cholesterol from brain is exported in the form of oxysterols, or metabolised to steroids, which in contrast to cholesterol can cross the blood-brain barrier. Steroids and oxysterols may be synthesised in brain, but can also be transported into brain from peripheral tissue. Both oxysterols and steroids have biological activity in brain. They can behave as ligands for classical nuclear receptors, and exert their effects over hours to days, or interact with neurotransmitter gated ion channels and modulate neural transmission exerting their effects in milliseconds. The exact sterol and steroid content of brain has yet to be thoroughly characterised. In this mini-review we will discuss mass spectrometry methods for the analysis of steroids and sterols in brain, and propose methods suitable for the profiling of different brain regions with high sensitivity (sub pg) and specificity.