This study explores the potential of profiling a microgram‐scale soft tissue biopsy by NMR spectroscopy. The important elements of high resolution and high sensitivity for the spectral data are achieved through a unique probe, HR‐μMAS, which allowed comprehensive profiling to be performed on microgram tissue for the first time under MAS conditions. Thorough spatially resolved metabolic maps were acquired across a coronal brain slice of rat C6 gliomas, which rendered the delineation of the tumor lesion. The results present a unique ex vivo NMR possibility to analyze tissue pathology that cannot be fully explored by the conventional approach, HR‐MAS and in vivo MRS. Aside from the capability of analyzing a small localized region to track its specific metabolism, it could also offer the possibility to carry out longitudinal investigations on live animals due to the feasibility of minimally invasive tissue excision.