In several neurological, psychiatric and developmental disorders there is an evidence of alternations in neurotransmitter concentrations in the brain,^{1, 2} especially in the most abundant excitatory and inhibitory neurotransmitters: Glutamate (Glu) and Gamma-aminobutyric acid (GABA), respectively.^{1, 3} The goal of this study was to estimate the measurement reproducibility of our fast and robust GABA-edited 3D-MRSI method and to validate its feasibility for (pre)clinical neurotransmitters studies.The study was performed on a 3T whole-body MR scanner (TIM Trio^®, Siemens Healthcare, Erlangen, Germany) using a 32-channel head coil (Siemens Healthcare, Erlangen, Germany) for signal reception. To ensure accurate and reproducible slice positioning we used auto-align and for identical VOI (80×85×60 mm³) placement 3D T₁-weighted MPRAGE images in three orthogonal directions. For spectroscopic measurements we applied a spiral-encoded GABA-edited MEGA-LASER 3D-MRSI sequence with real-time correction for motions, frequency, and scanner instability artefacts.^4,5 During GABA-editing we applied 60 Hz Gaussian pulses refocusing resonances at 1.9 ppm (EDIT-ON) and symmetrically at 7.5 ppm (EDIT-OFF) using 68 ms echo time. We achieved GABA⁺ (GABA plus macromolecule contamination) and Glx (Glu plus Glutamine contamination) 3D-mapping with ~3 cc nominal resolution in ~20 min. Fourteen healthy volunteers (age 30 ± 5 years; 7 men and 7 women) were scanned twice with a gap of ≤ 30 minutes for assessing the inter- and intra-subject variability. Measured MR data were finally processed employing LCModel, Matlab, Bash, and MINC software, and statistically evaluated using SPSS package.For spectral quantification were used two LCModel-fitted data sets, for EDIT-OFF and for DIFF (difference spectrum; subtraction of EDIT-ON and EDIT-OFF) spectrum (Fig. 1). Over all quantified voxels, we achieved FWHM and SNR mean values in the range of 7.2 - 10.1 Hz and 13.7 - 19.4, respectively (Fig. 2). In test-retest intra-subject variability evaluation we found low coefficient of variations (CV: mean of medians) and high intraclass correlation coefficients (ICC: mean) for GABA⁺ ratios (GABA⁺/tCr and GABA⁺/tNAA): CV ~8% / ICC >0.75, as well as Glx ratios (Glx/tCr and Glx/tNAA): CV ~6% / ICC >0.70. Likewise, there were low inter-subject median CV values for GABA⁺ ratios (~8%) as well as for Glx ratios (~6%). Finally, for all quantification-referencing combination the Bland-Altman plots of test-retest reproducibility investigations across the VOI in all fourteen subjects were displayed on Figure 3.Our spiral-encoding GABA-LASER sequence with real-time artefact correction enables fast metabolites 3D-mapping with minimal voxel size, high SNR, better localization accuracy, and high spectral quality. Furthermore, the intra- and inter-subject CV quantification for the repeated scans for GABA⁺ and Glx ratios in the voxel by voxel comparison across the whole VOI suggest that our GABA-edited 3D MRSI measurements were highly reproducible. A high degree of reliability between test-retest measurements was also confirmed using ICC analysis. Accordingly, our Bland-Altman plots showed the high 95% agreement of repeatedly measured GABA⁺ and Glx ratios.In vivo assessment of GABA⁺ and Glx levels using our spiral-encoded GABA-edited MEGA-LASER sequence provides highly reproducible 3D mapping of these neurotransmitters and thus can improve the understanding of several neurological as well as psychiatric disorders.