Maintaining the appropriate complement and content of lipids in cellular membranes is critical for normal neural function. Accumulating evidence suggests that even subtle perturbations in the lipid content of neurons and myelin can disrupt their function and may contribute to myelin and axonal degradation. In this study, we determined the composition and quantified the content of lipids and sterols in normal appearing white matter (NAWM) and normal appearing grey matter (NAGM) from control and multiple sclerosis brain tissues by electrospray ionization tandem mass spectrometry. Our results suggest that in active-multiple sclerosis, there is a shift in the lipid composition of NAWM and NAGM to a higher phospholipid and lower sphingolipid content. We found that this disturbance in lipid composition was reduced in NAGM but not in NAWM of inactive-multiple sclerosis. The pattern of disturbance in lipid composition suggests a metabolic defect that causes sphingolipids to be shuttled to phospholipid production. Modelling the biophysical consequence of this change in lipid composition of NAWM indicated an increase in the repulsive force between opposing bilayers that could explain decompaction and disruption of myelin structure.