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Bioluminescence imaging (BLI) is ubiquitous in scientific research for the sensitive tracking of biological processes in small animal models. However, due to the attenuation of visible light by tissue, and the limited set of near-infrared bioluminescent enzymes, BLI is largely restricted to monitoring single processes in vivo. Here we show, that by combining stabilised colour mutants of firefly luciferase (FLuc) with the luciferin (LH2) analogue infraluciferin (iLH2), near-infrared dual BLI can be achieved in vivo. The X-ray crystal structure of FLuc with a high-energy intermediate analogue, 5’-O-[N-(dehydroinfraluciferyl)sulfamoyl] adenosine (iDLSA) provides insight into the FLuc-iLH2 reaction leading to near-infrared light emission. The spectral characterisation and unmixing validation studies reported here established that iLH2 is superior to LH2 for the spectral unmixing of bioluminescent signals in vivo; which led to this novel near-infrared dual BLI system being applied to monitor both tumour burden and CAR T cell therapy within a systemically induced mouse tumour model., eLife digest Fireflies and some other insects glow to attract mates or prey. This so-called bioluminescence occurs when an enzyme called luciferase modifies the molecule luciferin, which can then emit bright yellow-green light. The gene that encodes the luciferase enzyme has been introduced into cells from mammals, including cancer cells. In the presence of luciferin, these cells begin to glow. The brightness of the bioluminescence depends on how many cancer cells are growing and dividing. The light is bright enough for the cancer cells making luciferase to be transplanted into mice so their behaviour can be examined. However, blood and other tissues absorb the yellow-green light, making it hard to see the cancer cells deep within a mouse. To circumvent this problem, researchers designed a new type of luciferin, called infraluciferin, which emits red light that shines through blood and tissues. There are now also different variants of the luciferase enzyme, which act on infraluciferin to make different shades of red light. Stowe et al. wanted to test if two different biological events happening at the same time could be observed using two shades of bioluminescent red in a single live mouse. First, a mixture of cancer cells containing two versions of luciferase were transplanted into mice. When the mice were then given infraluciferin, the two types of cancer cells could be distinguished based on the different shades of red bioluminescence. In a second experiment, Stowe et al. tracked the treatment of cancer cells with immune cells, by introducing a different version of luciferase into each of the two groups of cells. Over time, the red light produced by the immune cells grew stronger than that of the cancer cells, indicating that the number of cancer cells had decreased and that the treatment was effective. Together, this work shows that it can be simple, cheap and efficient to observe more than one cell type, or even disease, in a living system. This technique may be used by scientists to study different diseases and treatment options in mice. Importantly, it will also reduce the number of animals used to do this research.