Sensing hypoxia in tissues and cell models can provide insights into its role in disease states and cell development. Fluorescence imaging is a minimally-invasive method of visualising hypoxia in many biological systems. Here we present a series of improved bioreductive fluorescent sensors based on a nitro-naphthalimide structure, in which selectivity, photophysical properties, toxicity and cellular uptake are tuned through structural modifications. This new range of compounds provides improved probes for imaging and monitoring hypoxia, customised for a range of different applications. Studies in monolayers show the different reducing capabilities of hypoxia-resistant and non-resistant cell lines, and studies in tumour models show successful staining of the hypoxic region.