Observing cell nuclei in the 3D culture model was still challenging due to the limited penetration depth of lasers from the microscopes. Observation of deformation of cell nuclei is important because it is thought to modulate cellular functions. In this study, we have achieved optical clearing techniques for osteocytic spheroids using OptiPrep. Furthermore, we developed a novel 3D image analysis software to quantify the shape and position of the nuclei in the spheroids. As a result, we found out that the shape of cell nuclei was more sphere-shaped inside the spheroid. The combination of optical clearing and 3D image analysis techniques is expected to become powerful tools in the field of 3D culture models.