In ovo research is a valuable option of preclinical research, but imaging studies are severely limited by the costs of dedicated equipment needed for small-size eggs. We sought to verify the feasibility of using larger eggs of ostrich (Struthio camelus) for the imaging with PET/CT scanners used for routine clinical investigations. Methods: Ostrich eggs were incubated until shortly before hatching and prepared for the intra-vitelline venous injection of contrast media or radiotracers. Imaging was performed by native computed tomography (CT), contrast-enhanced CT and 18F-positron emission tomography/CT (PET/CT). Efforts were paid to identify the technical adaptations needed to improve the procedural outcomes. Results: Of the 34 eggs initially incubated, 12 became fully available for imaging of the embryonal development. In ovo imaging with conventional PET/CT was not only feasible, but it also provided images of good quality, including upon dynamic PET imaging. Conclusion: In ovo imaging with ostrich eggs and routine clinical scanners may allow a broader application of this field of preclinical research, bypassing the need of costly dedicated equipment and reducing the number of animals needed for classic animal research. Further experiments are warranted to refine this novel approach, in particular regarding reduction of motion artifacts and improvement of viability monitoring.