Targeted therapies such as PI3K inhibition can affect tumor vasculature, and hence delivery of imaging agents like FDG, while independently modifying intrinsic glucose demand. Therefore, it is important to identify whether perceived changes in glucose uptake are caused by vascular or true metabolic changes. This study sought to develop an optical strategy for quantifying tissue glucose uptake free of cross-talk from tracer delivery effects. Glucose uptake kinetics were measured using a fluorescent D-glucose derivative 2-(<i>N</i>-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-deoxy-Dglucose (2-NBDG), and 2-(<i>N</i>-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-deoxy-L-glucose (2-NBDLG) was used as a control to report on non-specific uptake. Vascular oxygenation (SO<sub>2</sub>) was calculated from wavelength-dependent hemoglobin absorption. We have previously shown that the rate of 2-NBDG delivery <i>in vivo</i> profoundly affects perceived demand. In this study, we investigated the potential of the ratio of 2-NBDG uptake to the rate of delivery (2-NBDG<sub>60</sub>/R<sub>D</sub>) to report on 2-NBDG demand <i>in vivo</i> free from confounding delivery effects. In normal murine tissue, we show that 2-NBDG<sub>60</sub>/R<sub>D</sub> can distinguish specific uptake from non-specific cell membrane binding, whereas fluorescence intensity alone cannot. The ratio 2-NBDG<sub>60</sub>/R<sub>D</sub> also correlates with blood glucose more strongly than 2-NBDG<sub>60</sub> does in normal murine tissue. Additionally, 2-NBDG<sub>60</sub>/R<sub>D</sub> can distinguish normal murine tissue from a murine metastatic tumor across a range of SO<sub>2</sub> values. The results presented here indicate that the ratio of 2-NBDG uptake to the rate of 2-NBDG delivery (2- NBDG<sub>60</sub>/R<sub>D</sub>) is superior to 2-NBDG intensity alone for quantifying changes in glucose demand.