Most solid tumors are hypoxic in nature due to the limited supply of oxygen to internal tissues. Hypoxia plays an important role in metabolic adaptations of tumorsÂ thatÂ contribute significantly to cancer pathogenesis. Among the several metabolic alterations induced by hypoxia, hypoxia-mediated increased glucose uptake serves as the hallmark of metabolic reprogramming. Hypoxia-mediated stabilization of hypoxia-inducible factor-1 alpha (HIF-1α) transcription factor leads to altered expression of several glycolytic genes and glucose transporters, which results in increased glucose uptake by tumor cells. Here we describe an easy and simple way of measuring the hypoxia-mediated tumor glucose uptake in vivo. The method is based on fluorescent imaging probe, RediJect 2-DG, which is a nonradioactive fluorescent-tagged glucose molecule. We have discussed orthotopic tumor implantation of HIF-1α knockdown and control pancreatic cancer cells and glucose uptake measurement in vivo by using IVIS imaging system along with reagent preparations.