<p>Osteosarcoma (OS) is a devastating primary bone cancer in dogs and humans. Histotripsy is a non-invasive ablation technique that mechanically destroys targeted tumor with focused ultrasound and has shown promise in treating small portions of canine OS followed by limb amputation. This study represents the first use of histotripsy to treat large volumes of canine OS, using fractionated treatments, without subsequent surgical resection. Our objective was to evaluate the safety and feasibility of treating large volumes of OS, following clinical outcomes after histotripsy. 9 dogs with suspected OS were treated with 1–5 histotripsy ablations, depending on tumor size, fractionated over 3 to 17 days. Pain, quality of life, and gait were followed over time, and MRI was used for pre-treatment planning and post-treatment monitoring. All dogs tolerated fractionated histotripsy treatments well, and the ablation zone was visible as a non-enhancing area on MRI matching the targeted location. There was a significant reduction in tumor contrast enhancement after completion of all histotripsy treatments and at the end of follow-up. Across all dogs with follow-up, there was a significant increase in pressure on the tumor-bearing limb during walking at the end of follow-up as compared to pre-treatment, indicating decreased pain. 4/6 dogs with follow-up showed a clinically significant decrease in pain. This study shows the promise of histotripsy as a non-invasive limb-sparing treatment for OS and advances the use of fractionated histotripsy treatments to ablate large and complicated tumors.</p>