In search of better predictive animal models for evaluating treatment response in lung cancer, orthotopic lung tumour models are a great step forward over traditional subcutaneous models. Crucial in the development of such orthotopic models is a reliable and reproducible instillation method. Because cells are instilled inside the thorax, the accuracy of the instillation and visualization of tumour growth demands the use of non-invasive imaging methods. We used a minimally invasive intratracheal intubation method to instill bioluminescent lung tumour cells in the lung parenchyma. Adaptation of the cell containing medium provides the possibility of tracing the exact location of the injection by means of single photon emission computed tomography/computed tomography (CT) imaging. The transplantation medium was also optimized to prevent migration of the injected substance. This results in the outgrowth of single and well-defined lung tumours at the instillation site. Finally, tumour growth was validated and longitudinally monitored with a combination of CT and bioluminescence imaging. The reported transplantation procedure enables the assessment of injection accuracy and provides a good approach for the generation of orthotopic lung tumour models for future response imaging studies.