Multiple sclerosis is a chronic inflammatory and neurodegenerative disease characterized by demyelination. To follow patients longitudinally and monitor treatment response, there is a need for robust and tissue-specific imaging biomarkers reflective of the heterogeneous disease course. Here, we aimed to validate REMyDI as an MRI-based measure of myelin ex vivo and in vivo. Histopathologically, REMyDI correlates well with all three of the studied myelin staining methods. In vivo, REMyDI revealed a strong sensitivity in differentiating white matter as compared to normal appearing white matter with associations to both cognitive (information processing speed) and physical disability (Expanded Disability Status Scale).