OBJECTIVES To describe a technique for transurethral tumour inoculation, bioluminescence imaging (BLI) and validation of this approach using ex vivo magnetic resonance imaging (MRI), as a reproducible and quantifiable model of orthotopic bladder cancer is required to enable preclinical pharmacological studies of intravesically administered anticancer agents and the use of BLI provides a sensitive method to monitor tumour growth over time. MATERIALS AND METHODS Human KU‐7 bladder tumour cells were transduced with a lentiviral construct to stably express the firefly luciferase gene. These cells were then inoculated in female nude mice by intravesical instillation. BLI was performed weekly and the mice were killed after 4 weeks. Ex vivo MRI and whole‐mount step‐sections were obtained to assess bladder tumour volume. RESULTS KU‐7 tumour cells were highly tumorigenic and were successfully inoculated in 96% of mice. After 4 weeks, all tumours were confined to the mucosa and submucosa (≤pT1). There was an excellent correlation between tumour volume and BLI for both ex vivo bladder MRI (R2 = 0.929) and end‐point histological measurements (R2 = 0.836). CONCLUSIONS We have established and validated a reliable model of orthotopic bladder cancer that can be used to evaluate various methods of intravesical therapy. BLI allows excellent longitudinal surveillance and quantification of tumour burden.