The development of a novel imaging mass spectrometry (IMS) method provides key information on the spatial distribution and quantification of pathogenic glycosphingolipids, hexosphingosines, and monohexosylceramides across the brain of the murine model of Krabbe disease, an inherited lysosomal deficiency of galactocerebrosidase, associated with a demyelinating disorder with a broad spectrum of age of onset. Here, GALC is linked to Krabbe disease.