Advances in fluorescence imaging coupled with the generation of near infrared probes have significantly improved the capabilities of non-invasive, real-time imaging in whole animals. In this study we were able to overcome a limitation of in vivo fluorescence imaging and have established a dual cell tracking method where two different cell types can be monitored according to the spectral signature of the cell labelling fluorophore. Using a mouse model of acute liver injury, we have characterised the in vivo migration patterns of wild type and transgenic neutrophils with impaired chemotaxis. Here, we were able to demonstrate that IVIS provides a sensitive multiplexing technology to differentiate two different cell populations based on the spectral signature of the cell labelling fluorophores. This spectral unmixing methodology has the potential to uncover multidimensional cellular interactions involved in many diseases such as fibrosis and cancer. In vivo spectral un-mixing provides a useful tool for monitoring multiple biological process in real-time in the same animal.