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Abstract Background Easily obtainable biomarkers for non-infectious, inflammatory central nervous system (CNS) disorders in dogs are lacking. Hypothesis/Objectives Dogs with non-infectious inflammatory CNS disorders might have specific neural autoantibodies in serum or cerebrospinal fluid (CSF), or both, discoverable by commercially available indirect immunofluorescence (IIF) assays. Animals Dogs (n = 23) with non-infectious inflammatory CNS disorders, including meningoencephalomyelitis of unknown origin, were recruited from the University Hospital for Companion Animals (Copenhagen, Denmark). Methods Using an exploratory, prospective study design, serum and CSF from dogs were investigated for specific neural autoantibodies with cell- and tissue-based IIF assays used to identify neural autoantibodies in humans. Results Astrocytic autoantibodies against glial fibrillary acidic protein were present in 2 dogs, neuronal targeted autoantibodies against N-methyl-D-aspartate receptors (NMDAR) in 2 dogs, and tripartite motif-containing protein 46 in 1 dog. Additionally, autoantibodies toward myelin were suspected in 6 dogs and myelin-oligodendrocyte glycoprotein in 4 dogs. Autoantibodies against a neuronal surface structure were suspected in 1 dog but could not be further specified. Conclusions and clinical importance This study confirmed the presence of neural autoantibodies in several dogs with non-infectious inflammatory CNS disorders. The importance of both tissue- and cell-based assays (CBA) is emphasized by the fact that all specific autoantibodies apart from those targeting NMDAR were detected by tissue-based assays and then confirmed on cell-based assays (CBA).