OBJECTIVE Increased lactate (Lac) level in brain tumours is in vivo detectable by 1H MR spectroscopy (MRS) but is frequently overlapped by strong lipid signals, which either leads to a weak quality of the Lac signal or even inhibit its detection. We sought to optimize the separation of Lac from lipid signals in intermediate-echo time MRS acquisitions thus allowing its applicability as clinical biomarker in glioblastomas. METHODS Data of 27 patients with glioblastoma multiforme (GBM) were analysed using standard post-processing software as well as in-house modified technique based on the same commercially available software. The patients' Lac concentration values provided by the MRS post-processing technique were converted to realistic concentrations by using an appropriately calibrated phantom. The Cramér-Rao lower bound (%CR) was the principal criterion for estimating the quality of the MRS post-processing results. RESULTS Based on the ex vivo calibration, the analysis of the in vivo MR spectroscopy measurements led to an improvement of the %CR(Lac) value from 18 % to 8 %. In a single case, the detection of Lac was achievable only by the modified technique, as Lac signal was contaminated with lipids using the standard analysis. The resulting in vivo Lac values from the modified analysis (median: 4.77 mmol/l, range: 1.5-9.2) were considered as a realistic order of magnitude for the metabolite concentrations, whereas no Lac was identified in the normal appearing white matter. This qualified also Lac mapping as a biomarker for regional heterogeneity in GBM. CONCLUSIONS The proposed methodology is a promising first step for more reliable analysis of Lac signal, decontaminating it from lipid peaks in MRS, and may help to establish Lac as a biomarker for brain tumours in clinical routine.