Although the in vivo metabolic pathways of scutellarin, a traditional Chinese medicine, have been investigated via different liquid chromatography techniques, studies on the distribution and location of scutellarin within organ tissue sections have not been reported. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can generate in situ spatial distribution profiles for scutellarin and its metabolites in a kidney section. However, the direct detection of a small molecule (m/z < 600) using conventional matrices often results in ion suppression and matrix interferences. In this study, we demonstrated a novel methodology using MALDI-MSI for the in situ spatial localization of scutellarin and its metabolites in kidney tissues by applying a binary matrix of graphene oxide (GO) and caffeic acid (CA). The results indicated that the binary matrix (GO/CA) significantly improved the detection efficiency of scutellarin and its metabolites with relatively high sensitivity, selectivity and reproducibility on tissue sections. This methodology was successfully applied to map scutellarin and its metabolites with MALDI-MSI in mouse kidney tissues. Specifically, scutellarin and scutellarein were found to be located in the cortex and medulla regions of the kidney with relatively high abundance, whereas the remaining metabolites appeared in the cortex with low abundance. We believe that the novel imaging methodology may also be used for the studies of cancerous tissues and inform the development of the future therapies of kidney tumors.