The study of late-stage human African trypanosomiasis (HAT) within mouse models is lengthy and complex, with the removal of brain tissue being required to monitor parasitic burden. This results in the inability to track real-time infections within the central nervous system (CNS). With nearly 70 million people at risk of HAT infection in Africa every year, research into new drug therapies which are capable of crossing the blood brain barrier and efficacious towards the parasite are imperative. However, progress has been slow and difficult, partly due to the limitations with the current drug relapse mouse model for African trypanosomiasis (T. b. brucei GVR35), which requires a follow-up time of 180-days. In this study, we report the generation of highly bioluminescent parasites and their use in an in vivo imaging model of late-stage African trypanosomiasis. Bloodstream forms of the chronic model strain GVR35 were transfected with a “red-shifted” luciferase, which produced detectable signal in CNS at 21-days p.i mimicking that of the wild type line. This model enabled the tracking of a single animal through the entire chronic infection, with the detection of parasites occurring earlier than blood film microscopy. The model was further employed to assess the effects of known anti-trypanosomal drugs on bioluminescence, and to demonstrate how the reduction in bioluminescent signal combined with qPCR can determine a dose-dependent effect after treatment. The non-invasive in vivo imaging model will reduce the time and numbers of mice required to assess preclinical efficacy of new anti-trypanosomal drugs. This study shows the development and optimisation of a new, efficient method to evaluate novel anti-trypanosomal drugs in vivo with the added advantage of reducing the current drug relapse model from 180-days to 90-days.