Up to 30% of patients with temporal lobe epilepsy (TLE) remain without remarkable changes in MRI. In this study we investigated the role of 1H-MR spectroscopy (1H-MRS) in lateralizing the affected hemisphere in the mentioned patient group. Twenty-two consecutive patients diagnosed with TLE were investigated by high resolution MRI and 1H-MRS. We examined the incidence and diagnostic accuracy of temporal metabolite alterations determined by Linear Combination of Model Spectra (LCModel) via water reference. Metabolite values of each hemisphere of TLE patients were compared with healthy controls. Results of metabolite alterations were related to intensive video EEG focus localization. Reduction of N-acetylaspartate + N-acetylaspartyl-glutamate (tNAA) in the affected hemisphere revealed identification in six of nine patients (66%) with unilateral TLE. Group comparison revealed a significant reduction of tNAA (6.1 ± 0.8*) in the involved temporal lobe compared with controls (6.67 ± 0.4*, P = 0.026). Choline levels were significantly increased in the affected hemisphere (1.42 ± 0.17*) compared with healthy controls (1.22 ± 0.17*, P = 0.035). The results of our study show that 1H-MRS is able to identify the affected hemisphere of MRI negative TLE patients and can be used as an additive tool in multimodal focus localization.