To evaluate the diagnostic performance of 2-hydroxyglutarate magnetic resonance spectroscopy (2HG-MRS) for non-invasive detection of isocitrate dehydrogenase (IDH) mutations in gliomas and to assess its clinical utility in a routine setting, we conducted a retrospective single-center study. A total of 135 patients with suspected gliomas underwent MRS, of whom 81 had resection and histopathological confirmation. All patients received single-voxel 2HG-MRS using a PRESS sequence on a 3T MRI scanner. A positive MRS result was defined as either an absolute 2HG concentration ≥1.0 mM with fitting error ≤15%, or a 2HG/creatine (2HG/Cr) ratio ≥0.252. Sensitivity, specificity, and predictive values were calculated, and ROC curve analysis compared both quantification strategies. Technical and anatomical sources of error, such as voxel placement near cerebrospinal fluid (CSF) or resection cavities, were also documented. Absolute 2HG quantification showed a sensitivity of 36.6% for detecting IDH-mutant gliomas, with a specificity of 100% for excluding IDH-wildtype glioblastomas. Using the 2HG/Cr ratio ≥0.252 improved sensitivity to 75.6%, though specificity declined to 75.0%. Among WHO grade 4 IDH-mutant astrocytomas, sensitivity increased to 71.4% using absolute 2HG and 85.7% with the ratio method. ROC analysis demonstrated superior accuracy for the 2HG/Cr ratio method compared to absolute quantification. All misclassified cases were attributed to technical or anatomical limitations such as voxel misplacement. Six discordant cases were validated by LC-MS/MS on frozen tumor tissue, confirming acquisition-related misclassification rather than metabolic overlap. In conclusion, while 2HG-MRS shows promise as a non-invasive tool for IDH mutation detection, it lacks the reliability required for universal application across all glioma cases. It performs better in larger lesions and pre-resection settings but is limited by susceptibility to significant acquisition errors. Additional diagnostic modalities are needed to improve prediction accuracy for IDH mutation status in clinical practice.