Background: Non-invasive diagnosis of pediatric brain tumors can be challenging due to diverse tumor pathologies and similar imaging appearances. Magnetic resonance spectroscopy (MRS), when combined with high spatial resolution anatomic imaging obtained with conventional magnetic resonance imaging (MRI), provides metabolic information within the lesion as well as the surrounding tissue. The differentiation of neoplastic from non-neoplastic lesions and low-grade from high-grade neoplasms is essential for determining the choice of treatment and the best treatment plan. We aimed to measure specific metabolic ratios and evaluate metabolic profiles of various lesions by MRS to assist in making a more confident diagnosis. Results: The choline/creatine (Cho/Cr), choline/N-acetylaspartate (Cho/NAA), and Cho/NAA+Cr ratios all had statistically significant values for the differentiation between neoplastic and non-neoplastic lesions at cutoffs 1.8, 2, and 0.8 respectively. The Cho/NAA, Cho/Cr, Cho/NAA+Cr, and myo-inositol/creatine (mI/Cr) ratios all had statistically significant values for the differentiation of high-grade from low-grade neoplasms at cutoffs 3.3, 3.5, 1.3, and 1.5 respectively. The presence of a lipid lactate peak was only significant for differentiating high-grade from low-grade neoplasms. Medulloblastomas, diffuse pontine gliomas, and choroid plexus carcinoma all showed characteristic metabolic profiles on MRS. Metastasis showed lower Cho/ NAA and Cho/Cr ratios outside the tumor margin than high-grade neoplasms. Conclusion: The use of certain metabolite ratios with high sensitivity and specificity to distinguish neoplastic from nonneoplastic lesions and low-grade from high-grade neoplasms while assessing the metabolic profile of the lesion aids in the non-invasive diagnosis of pediatric brain tumors. MRS facilitates earlier treatment planning by determining tumor spatial extent and predicting tumor behavior with potential to solve sampling problems of inaccessible and heterogenous lesions as well as unnecessary sampling of benign lesions.