Detecting glutamate signals in vivo within the brain may have a diagnostic potential. Recently, with many translational research efforts leading to clinical trials, presenting the normal in vivo glutamate distribution in pre-clinical data to establish a database for pre-clinical studies can be valuable. In this abstract, we investigated glutamate signal distributions in multiple brain regions of a healthy rat brain using GluCEST imaging. Quantified GluCEST signals showed significant differences between white and gray matter regions. Our findings and investigations yield a valuable database and insights for comparing glutamate signal changes in pre-clinical brain diseases.