Here we report a complementary approach, based on the 4,5 indolequinone scaffold, to develop hypoxia-sensing probes that detect different levels of hypoxia in 2D and 3D cell culture. We show that the indolequinone groups are readily amenable to chemical modification through ether bond formation with the commonly used fluorophores 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl (DDAO), resorufin, and Me-Tokyo green, affording a range of hypoxia-sensing fluorescent probes 1-4 (Figure ). We demonstrate that these probes are enzymatically reduced, in an oxygen dependent manner, to reveal a palette of fluorophores with emission maxima ranging through red, orange, and green. Varying the pKa of the dye and the substitution on the methylene unit of the indolequinone, results in compounds that are activated at different oxygen concentrations, ranging from ≤6%, ≤4%, ≤2%, and ≤1 %, and at different rates. Validation of these probes in 2D and 3D cell culture demonstrates that they are powerful and convenient tools for sensing a range of different hypoxia levels.