Bioluminescent imaging is a powerful technique that enables imaging in living organisms with high sensitivity, low background signal, low cost and without the need for radioactivity. The emitted photons are produced in the oxidation reaction of luciferin catalyzed by luciferase. In the caged luciferin approach, the bioluminescent readout is used for measuring the activity of a specific biochemical process. Luciferin is caged in order to prevent catalysis by luciferase, and only when the cage is removed by a specific biochemical event of interest, luciferin is released and becomes a substrate of luciferase. Hence, emitted photons are proportional to the amount of free luciferin, thus allowing to monitor the dynamics of the investigated process. Recent progress in the development of bioorthogonal reactions enabled to answer several fundamental biological questions, improve clinical diagnosis, and to better evaluate therapy efficacy. For example, one of the most robust biorthogonal reactions is the Staudinger ligation, which occurs between a properly tuned azide and a phosphine. In this work, we have used the caged luciferin approach combined with the Staudinger ligation to monitor metabolite uptake in living animals. We have successfully applied this method for the monitoring uptake of two metabolites â glucose and nicotinamide riboside (NR). Glucose is a major source of energy for most living organisms and its aberrant uptake is linked to many pathological conditions. On the other hand, NR is a form of vitamin B3 and represents one of the salvageable NAD+ precursors. Low NAD+ level inside of cells is strongly linked to numerous metabolic and age-related disorders. Despite of their importance, in both cases our understanding of disease-associated glucose or NR flux is limited due to a lack of robust tools. To date, positron emission tomography (PET) imaging remains the gold standard for measuring glucose uptake and no optical tools exist for non-invasive longitudinal imaging of neither of the two metabolites in in vivo settings. Here we report the development of a novel bioluminescent glucose uptake probe (BiGluc) and bioluminescent NR uptake probe (BiNR) for real-time, non-invasive longitudinal imaging of glucose and NR absorption both in vitro and in vivo. The new imaging reagents enable a wide range of applications in the field of metabolism and drug development.