Objectives Magnetic resonance spectroscopy is a non-invasive technique for probing metabolism and underpins advanced methods such as magnetic resonance spectroscopic imaging (MRSI) and diffusion-weighted spectroscopy (DWS). MRSI enables spatial mapping of metabolite distributions, offering insights into regional metabolic heterogeneity that single-voxel spectroscopy (SVS) cannot capture. However, MRSI produces large multidimensional datasets and requires complex processing pipelines, limiting reproducibility and accessibility. While human studies benefit from advanced processing tools, similar developments in preclinical research remain scarce, highlighting a demand for practical tools accessible to non-experts. Methods To address this need, we introduce the MRS4Brain Toolbox , a freely available MATLAB-based platform for preclinical spectroscopy, including MRSI, SVS, and DWS. Results The toolbox integrates reconstruction, preprocessing, quantification, quality control, brain segmentation automatically overlaid on metabolite maps, modeling, and statistical analysis into unified workflows accessible via a graphical interface. Conclusion By streamlining data processing and reducing technical barriers, MRS4Brain Toolbox promotes reproducibility, harmonization, and broader adoption of advanced spectroscopic techniques in preclinical studies, ultimately facilitating translational research.