Glutamate excitotoxicity plays a key role in the etiology of a variety of neurological, psychiatric, and neurodegenerative disorders. The goal of this study was to investigate spatiotemporal distribution in the brain and cerebrospinal fluid (CSF) concentrations of ubiquitin C-terminal hydrolase-1 (UCH-L1), glial fibrillary acidic protein (GFAP), αII-spectrin breakdown products (SBDP150, SBDP145, and SBDP120), and their relationship to neuropathology in an animal model of kainic acid (KA) excitotoxicity. Triple fluorescent labeling and Fluoro-Jade C staining revealed a reactive gliosis in brain and specific localization of degenerating neurons in hippocampus and entorhinal cortex of KA-treated rats. Immunohistochemistry showed upregulation of GFAP expression in hippocampus and cortex beginning 24h post KA injection and peaking at 48h. At these time points concurrent with extensive neurodegeneration all SBDPs were observed throughout the brain. At 24h post KA injection, a loss of structural integrity was observed in cellular distribution of UCH-L1 that correlated with an increase in immunopositive material in the extracellular matrix. CSF levels of UCH-L1, GFAP, and SBDPs were significantly increased in KA-treated animals compared with controls. The temporal increase in CSF biomarkers correlated with brain tissue distribution and neurodegeneration. This study provided evidence supporting the use of CSF levels of glial and neuronal protein biomarkers to assess neurotoxic damage in preclinical animal models that could prove potentially translational to the clinic. The molecular nature of these biomarkers can provide critical information on the underlying mechanisms of neurotoxicity that might facilitate the development of novel drugs and allow physicians to monitor drug safety.