In summary, we have developed an operationally simple protocol to prepare a palette of hypoxia-sensing fluorescent probes (1-4) with a range of emission colors though red, orange, and green. These probes are activated reductively under either chemical or enzymatic conditions to release their fluorescent reporters. The rates at which the fluorescent reporters are released correlate with the pKa of the reporters and are accelerated by the addition of a benzylic methyl group on the indole quinone bioreductive group. Compound 1 displayed unexpected redox properties resulting from the parent dye DDAO undergoing enzymatic reduction in hypoxia. However, the reduced form of DDAO ( ) can be re-oxidized when exposed to 21% O2 meaning that this probe is useful for detecting mild hypoxia in certain settings. The reversibility displayed by DDAO could form the basis for the design of more dynamic dyes capable of imaging fluctuations in hypoxia in real time. The resorufin-based probe 2 was activated in conditions of 4% O2 and lower, while the Tokyo green-based probe 4 was only activated in severe hypoxia -0.5% O2 and less. Application of these compounds in spheroids revealed that compound 2 images similar levels of hypoxia to pimonidazole, while compound 4 images more extreme hypoxia in a manner analogous to EF5. Therefore, compounds 2 and 4 represent convenient small molecule alternatives to image hypoxia in cells and spheroids without the need for secondary detection steps involving immunostaining. Most importantly, probes 2 and 4 could be used in combination to enable two color imaging of the heterogeneous hypoxic environments within spheroids.