Survival and growth of cellular grafts in tissue engineering (TE) are limited by the rate of oxygen (O2) and nutrient diffusion. As such, monitoring the levels of nutrients and O2 available to the cells is essential to assess the physiology of the cells and to evaluate strategies aiming at improving nutrient availability. In this article, a reporter system containing the luciferase gene driven by a hypoxia responsive promoter was used to monitor cellular hypoxia in a TE context. We report that luciferase activity correlates with the O2 tension in the cell culture medium. When transgenic cells were seeded onto scaffolds and implanted in immune-deficient mice subcutaneously, luciferase activity was detected. To validate the response to O2 levels of this reporter system, we cultured transgenic cells on biomaterials in a flow perfusion bioreactor and observed that cells in the bioreactor displayed a drastically lower luciferase activity than conventional static culture, and that higher luciferase activity is observed in the interior of a tissue-engineered construct, illustrating the uneven O2 distribution in three-dimensional constructs under conventional static culture. We conclude that this reporter system is a versatile tool to investigate cellular O2 availability in TE both in vitro and in vivo.