Background: High grade gliomas (HGG) are diffusely infiltrative brain tumours with dismal prognosis. Recent studies from our lab have demonstrated that glioma cells form synapses with surrounding neurons, and proliferate in response to neuronal input. How these neuron-glioma networks develop, and are influenced by experience, is currently unknown. We aimed to develop a novel imaging tool to study neuron-glioma cell interactions in freely behaving animals. Methods: Several patient-derived HGG cell lines were transfected to express the green calcium indicator GCaMP6s. These cells were xenografted into the premotor cortex of mice, along with a virus expressing the red calcium indicator jRGECO1a under a neuron-specific synapsin promotor to allow dual-color imaging of neurons and glioma cells. The Inscopix mini-microscope system was implanted into the cortex to allow real-time live calcium imaging in freely behaving animals. Results: Several HGG cell lines effectively expressed the GCaMP6s calcium indicator. In vivo, we were successfully able to image both neurons and glioma cells simultaneously in freely behaving mice in real time. Conclusions: The Inscopix system has been modified for studying cancer cells for the first time. This technology will be used to study how pharmacological agents and neuronal experience shape neuron-glioma circuit dynamics, to develop new therapeutic strategies for HGG.