In recent years, significant breakthroughs have been made in using in vitro tumor cell aggregates as a model to simulate the in vivo tissue environment. This technology enables discrete cells planted in low adsorption circular bottom well plates to aggregate into spheres. And spheres are believed to be able to simulate tumor behavior more effectively than conventional two-dimensional (2D) cell cultures. Because similar to tumor entities, 3D cell spheres also contain cells exposed on a spherical surface and cells deeply buried within the sphere, both containing proliferating and non proliferating cells, with a hypoxic environment at the center of the sphere. More and more examples have successfully used this three-dimensional spherical model for compound screening in various applications to study potential cancer therapies. However, obtaining a reliable method for analyzing such 3D spheres is challenging: focusing on a single field of view allows for the complete sphere to be seen, enabling batch imaging of each hole. Optimize the compound and dyeing process to ensure dye penetration and avoid interfering with the position of the sphere. Obtain representative images throughout the entire 3D structure to minimize defocusing and background signals on the imaging plane. Quickly analyze images to generate meaningful results, from which experimental conclusions can be drawn.