Canine osteosarcoma (OSA) is a malignant cancer with a poor prognosis. Cytokines mediate communication between bone marrow cells, including OSA cells when present. Understanding the role of cytokines in cancer may uncover their potential as prognostic indicators, and/or uncover novel therapeutic targets, helping to guide treatment. This study investigated serum cytokine profiles of dogs with OSA compared to healthy dogs and in relation to overall survival. Serum samples were collected from 63 appendicular OSA canine patients at time of diagnosis, 19 of these OSA patients throughout standard of care (SOC) treatment, and 19 healthy dogs. Cytokine profiles obtained via Multiplex Assay quantified 13 analytes. Results showed IL-8 and IP-10 were significantly increased in OSA patients compared to healthy dogs. IP-10 significantly decreased throughout SOC treatment. Higher IL-8 and IP-10 concentrations significantly associated with shorter survival time. These results suggest IL-8 and IP-10 may have prognostic value for canine OSA patients.