Osteosarcoma, a common malignancy in large dog breeds, typically metastasises from long bones to lungs and is usually fatal within 1 to 2 years of diagnosis. Better therapies are needed for canine patients and their human counterparts, a third of whom die within 5 years of diagnosis. We compared the in vitro sensitivity of canine osteosarcoma cells derived from 4 tumours to the currently used chemotherapy drugs doxorubicin and carboplatin, and 4 new anti-cancer drugs. Agents targeting histone deacetylases or PARP were ineffective. Two of the 4 cell lines were somewhat sensitive to the BH3-mimetic navitoclax. The proteasome inhibitor bortezomib potently induced caspase-dependent apoptosis, at concentrations substantially lower than levels detected in the bones and lungs of treated rodents. Co-treatment with bortezomib and either doxorubicin or carboplatin was more toxic to canine osteosarcoma cells than each agent alone. Newer proteasome inhibitors carfilzomib, ixazomib, oprozomib and delanzomib manifested similar activities to bortezomib. Human osteosarcoma cells were as sensitive to bortezomib as the canine cells, but slightly less sensitive to the newer drugs. Human osteoblasts were less sensitive to proteasome inhibition than osteosarcoma cells, but physiologically relevant concentrations were toxic. Such toxicity, if replicated in vivo, may impair bone growth and strength in adolescent human osteosarcoma patients, but may be tolerated by canine patients, which are usually diagnosed later in life. Proteasome inhibitors such as bortezomib may be useful for treating canine osteosarcoma, and ultimately may improve outcomes for human patients if their osteoblasts survive exposure in vivo, or if osteoblast toxicity can be managed.