Brassinosteroids (BRs) are a group of polyhydroxylated plant steroidal hormones crucial for many aspects of a plant’s life. Although it has been known that the biosynthesis of and signaling responses to BRs vary across cells and tissues, developmental stages and environmental conditions, the spatiotemporal distribution of BRs in vivo remains unresolved, owing to the lack of investigation tools. Moreover, whereas BRs do not travel by a long-distance transport, whether BRs can undergo short-distance transport, or BRs synthesized in one cell might traverse short distances to the neighboring cells, remains elusive. Here, we propose to investigate the spatial localization and abundance of endogenous BRs in Arabidopsis roots at the cellular level by mass spectrometry imaging (MSI). Furthermore, we plan to unravel the intercellular short-distance transport of BRs in roots by MSI with the aid of transgenic tools capable of modulating BR trafficking in a controlled manner. In addition, we intend to identify BR binding proteins, which may lead to the discovery of long-sought BR transporters, by limited proteolysis-coupled mass spectrometry (LiP-MS). This is an interdisciplinary project involving a number of collaborations with experts in their fields. We believe that this research will provide a powerful way to monitor BR distribution in different tissues and cells, generate for the first time a high-resolution map of cellular BR distribution in plants, and address the long-standing mystery of BR transport. The knowledge obtained here will be the foundation for further understanding of the mechanisms underlying the spatiotemporal regulation of BR signaling and physiological effects, which can be translated for crop genetic improvement.