Osteosarcoma is an aggressive cancer that often metastasizes early, presenting challenges for treatment. Traditional approaches like surgery and chemotherapy offer limited success for non-surgical cases. Spatially Fractionated Radiation Therapy (SFRT) is a promising strategy that delivers high-dose radiation selectively to tumor regions, minimizing damage to surrounding healthy tissues and providing a novel approach to limb preserving treatments, especially in cases where surgery is not feasible. This study leverages the potential of SFRT to treat large, radio-resistant tumors like osteosarcoma more effectively. Previous studies have demonstrated the high clinical efficacy of SFRT in other cancers. The combination of chemotherapy and gold nanoparticles (GNPs) remains underexplored, offering an exciting opportunity to enhance therapeutic effects while minimizing toxicity. This study explores the combination of SFRT with chemotherapy (CHEMO) and GNPs to treat canine osteosarcomas. The central hypothesis is that low-dose SFRT combined with CHEMO and/or GNPs can achieve a higher therapeutic ratio than traditional open-field radiation therapy with the same agents in vitro. The experimental design involves testing the effects of SFRT on mesenchymal stem cells and two osteosarcoma cell lines. Specific aims include characterizing the response of healthy cells and cancer cells to SFRT and CHEMO and investigating the multi-modality treatment approach using SFRT with GNPs or CHEMO. Experiments will be conducted using widely available radiation oncology equipment and cell colony survival assays, and the results will be evaluated by comparing therapeutic ratios to assess the effectiveness of the treatments. If successful, this study could inform new treatment strategies for aggressive osteosarcoma cases, combining radiosensitizers and novel radiation techniques for better outcomes and laying the foundation for clinical trials.