Abstract Background Equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy (eNAD/EDM) is an inherited neurodegenerative disorder associated with a vitamin E deficiency within the first year of life. Vitamin E consists of 8 isoforms metabolized by the CYP4F2 enzyme. No antemortem diagnostic test currently exists for eNAD/EDM. Hypothesis/Objectives Based on the association of α‐tocopherol deficiency with the development of eNAD/EDM, we hypothesized that the rate of α‐tocopherol, but not γ‐tocopherol or tocotrienol metabolism, would be increased in eNAD/EDM‐affected horses. Animals Vitamin E metabolism: Proof of concept (POC) study; eNAD/EDM‐affected (n = 5) and control (n = 6) horses. Validation study: eNAD/EDM‐affected Quarter Horses (QHs; n = 6), cervical vertebral compressive myelopathy affected (n = 6) horses and control (n = 29) horses. CYP4F2 expression and copy number: eNAD/EDM‐affected (n = 12) and age‐ and sex‐matched control (n = 11‐12) horses. Methods The rates of α‐tocopherol/tocotrienol and γ‐tocopherol/tocotrienol metabolism were assessed in equine serum (POC and validation) and urine (POC only) using liquid chromatography tandem mass spectrometry (LC‐MS/MS). Quantitative reverse‐transcriptase PCR (qRT‐PCR) and droplet digital (dd)‐PCR were used to assay expression and genomic copy number of a CYP4F2 equine ortholog. Results Metabolic rate of α‐tocopherol was increased in eNAD/EDM horses (POC,P