Glucose is the main energy source for cells in an organism and its blood concentration is tightly regulated in healthy individuals. However, impaired blood glucose control has been found in diseases such as metabolic syndrome and diabetes, and anomalous glucose utilization in cancer tissues. Dissecting the dynamics of the different phenomena involved in glucose handling (extracellular mass transport, membrane diffusion, and intracellular phosphorylation) is very relevant to identify which mechanisms are disrupted under disease conditions. In this work, we developed an effective methodology for quantitatively analyzing these phenomena in living cells. A measurement of steady-state glucose uptake is, by itself, insufficient to determine the dynamics of intracellular glucose. For this purpose, we integrated two types of measurements: cytosolic glucose concentration at the single-cell level, obtained using a cytosolic FRET nanosensor, and cell population glucose uptake, obtained without perturbing culture conditions using a microfluidic perfusion system. Microfluidics enabled accurate temporal stimulation of cells through cyclic pulses of glucose concentration at defined flow rates. We found that both, glucose uptake and phosphorylation, are linearly dependent on glucose concentration in the physiological range. Mathematical modeling enabled precise determination of the kinetic constants of membrane transport (0.27 s(-1)) and intracellular phosphorylation (2.01 s(-1)).