Abstract: MALDI-MS is an emerging technique that enables rapid profiling of endogenous substances in a high throughput. Nevertheless, its application to metabolite analysis is often hindered by the presence of massive matrix background peaks using conventional matrices. Herein rhein was introduced as a novel matrix for MALDI-MS analysis of cation metabolites. Several strong and weak basic metabolites were measured using rhein. Compared to the previously reported ionless matrix NSA and conventional matrix CHCA, clean MALDI-MS spectra were obtained devoid of matrix signals. Subsequently, we applied this matrix to profile a biologically complex sample, mice intestinal contents. In addition, we have validated the applicability of rhein to MALDI-MS imaging, which allows for simultaneous mapping of hundreds of metabolites from single mouse ileum section. In summary, the discovery of rhein as a novel MALDI matrix enables efficient and reliable profiling and imaging of endogenous metabolites from biological samples with no matrix interferences, which will greatly promote the utility of MALDI-MS-based platform for metabolome studies.