The direct link between sugar uptake and metabolic diseases highlights the iminent need for molecular tools to detect and evaluate alterations in sugar uptake efficiency as approaches to identify disease-relevant metabolic alterations. However, the strict requirements of facilitative glucose transporters regarding substrate binding and translocation pose challenges for developing effective fluorescence molecular probes. Based on the state-of-the-art understanding of glucose recognition by facilitative transporters (GLUTs), we designed a glucopyranoside mimic - GluRho - that delivers the "turn-on" rhodamine B to live cells glucose transport, including major transporters GLUTs 1-4. The high binding affinity achieved through the secondary interaction between the fluorophore and a GLUT protein supports the delivery of the probe in nutrient-rich conditions, facilitating its use as a tool for a direct assessment of glucose GLUT activity in live cells and organisms and across various experimental settings, including uptake evaluation in the presence of sugars or GLUT activity modulators. The lack of metabolic contribution to the probe uptake due to the elimination of the phosphorylation site contributes to the high efficacy of the GluRho probe in reflecting alterations in glucose uptake efficiency in live cells, between cell lines, and in multicellular model organisms, such as . The molecular modeling analysis of GluRho complexes with GLUT1 and GLUT2 provided essential information on GLUT-probe interactions, highlighting the residues facilitating the effective binding and translocation of the probe through transporters, thus setting the basis for developing glucose-based glycoconjugates as a cargo-delivering platform.