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Simple Summary Osteosarcoma is a bone cancer with 75% of cases occurring in people younger than 25 years old. 35–45% of patients demonstrate resistance to chemotherapeutics and critically, survival rates for osteosarcoma is only 10–30% for patients with metastases. Therefore, reliable and patient-specific drug testing modalities are needed. Organotypic slice culture consists of sections of tumours, which survive and preserve the tumours mechanical and cellular properties, thereby enabling personalised testing of drugs. This study aimed to characterise organotypic slice cultures of osteosarcoma bone tumours derived from mice and dogs and to use these models for testing of anti-tumoural drugs. This study reports the various cell constituents of the model and the maintenance of osteosarcoma organotypic cultures over several weeks. A significantly decreased sensitivity to chemotherapy in 3D organotypic culture relative to 2D monolayer was found, highlighting the need to test anti-cancer drugs in a more personalized and biomimetic manner. Abstract Improvements in the clinical outcome of osteosarcoma have plateaued in recent decades with poor translation between preclinical testing and clinical efficacy. Organotypic cultures retain key features of patient tumours, such as a myriad of cell types organized within an extracellular matrix, thereby presenting a more realistic and personalised screening of chemotherapeutic agents ex vivo. To test this concept for the first time in osteosarcoma, murine and canine osteosarcoma organotypic models were maintained for up to 21 days and in-depth analysis identified proportions of immune and stromal cells present at levels comparable to that reported in vivo in the literature. Cytotoxicity testing of a range of chemotherapeutic drugs (mafosfamide, cisplatin, methotrexate, etoposide, and doxorubicin) on murine organotypic culture ex vivo found limited response to treatment, with immune and stromal cells demonstrating enhanced survival over the global tumour cell population. Furthermore, significantly decreased sensitivity to a range of chemotherapeutics in 3D organotypic culture relative to 2D monolayer was observed, with subsequent investigation confirming reduced sensitivity in 3D than in 2D, even at equivalent levels of drug uptake. Finally, as proof of concept for the application of this model to personalised drug screening, chemotherapy testing with doxorubicin was performed on biopsies obtained from canine osteosarcoma patients. Together, this study highlights the importance of recapitulating the 3D tumour multicellular microenvironment to better predict drug response and provides evidence for the utility and possibilities of organotypic culture for enhanced preclinical selection and evaluation of chemotherapeutics targeting osteosarcoma.