Despite its essential role in the (patho)physiology of several diseases, CB₂R tissue expression profiles and signaling mechanisms are not yet fully understood. We report the development of a highly potent, fluorescent CB₂R agonist probe employing structure-based reverse design. It commences with a highly potent, preclinically validated ligand, which is conjugated to a silicon-rhodamine fluorophore, enabling cell permeability. The probe is the first to preserve interspecies affinity and selectivity for both mouse and human CB₂R. Extensive cross-validation (FACS, TR-FRET and confocal microscopy) set the stage for CB₂R detection in endogenously expressing living cells along with zebrafish larvae. Together, these findings will benefit clinical translatability of CB₂R based drugs. For more information please read the full publication DOI: 10.1039/D1SC06659E here.