To improve spatial resolution in in vivo bioluminescence imaging, a photon scattering correction, image restoration method was tested. The chosen algorithm was tested on in vivo bioluminescent images acquired on three representative tumor models: subcutaneous, pulmonary, and disseminated peritoneal. Tumor size was chosen as a quantitative criterion, such that the tumor reference measurements (determined photographically or by computed tomography) were compared to those derived from bioluminescent images, before and after restoration. This technique allowed a significant reduction to be achieved in the relative error between reference measurements and dimensions derived from bioluminescent images. In addition, improved delineation of the tumor foci was achieved. The restoration method allows spatial resolution in bioluminescence imaging to be improved, with interesting perspectives in terms of staging and quantitation in experimental oncology.