Spheroids are three-dimensional cell clusters that serve as reliable in vitro models for cancer drug screening, mimicking tumor microarchitecture and chemoresistance. Despite their potential, current spheroid culture systems lack essential but highly challenging features, such as automatic real-time imaging, treatment response assessment, and detailed live image capturing without disturbing the spheroid structure. To address these challenges, we developed a custom culture dish with a micro-patterned agarose structure, fabricated from a 3D-printed mold. This innovative tool facilitates spheroid growth and immobilization, enabling automated high-throughput imaging and data collection. It allows the microscope objective to approach the spheroid closely (within micrometers) while it floats in the culture medium, in a mapped position. Furthermore, the platform is compatible with several imaging systems, including standard, confocal and dual-photon microscopy. We successfully demonstrated the effectiveness of our platform by culturing and treating osteosarcoma spheroids with different concentrations of a standard chemotherapeutic agent and by capturing confocal images of extracellular matrix antigens in live spheroids. Additionally, we showed that the platform is compatible with viability and metabolic assays (e.g. Alamar blue and acid phosphatase), with minimal reagent consumption and cost-effective, simultaneous imaging-based assays. In conclusion, we propose an innovative platform for the study of tumor spheroids and other three-dimensional cellular structures, which allows tracking of size, shape, antigen expression, viability, and indirect monitoring of cell count over time. This advancement enhances our capacity to conduct in-depth investigations of cell behavior and therapeutic responses, contributing significantly to cancer research and drug development.