Osteosarcoma (OS) is the most common primary malignant bone tumour in children and adolescents. Therapy today includes surgical removal of the tumour and neoadjuvant and adjuvant chemotherapy. The 5-year survival rates for patients with localised disease are between 50 and 70%, but in patients with metastases the prognosis remains poor (∼20%). The aim of this study was the development of a biological relevant OS 3D microtissue model, which is suitable for drug development. Microtissues were formed by the hanging drop method with the established OS cell lines SaOS-2, HOS and MG-63, as well as with cells derived from osteoblastic and chondroblastic OS patient material. Histological characterisation of the microtissues with H/E- and Ki-67-(proliferation), as well as apoptosis staining (TUNEL) revealed the inherent histological heterogeneity of OS. Microtissues from SaOS-2 and HOS cell lines were exposed to doxorubicin, cisplatin, taurolidine, pemetrexed and taxol and the viability was assessed by the CellTiter-GLO(®) Luminescent Cell Viability Assay. The obtained IC50-values for 3D cultures were all higher (1.7 to >16,000-fold) when compared to corresponding cells grown in 2D monolayer culture, except for pemetrexed that was inactive in 2D and 3D cultures. Doxorubicin did not affect the viability of chondroblastic monolayer cultures whereas on 3D microtissues an IC50-value of 2.3μM was obtained. The 3D microtissues reflect the tissue heterogeneity of OS and are potential suitable tools for drug development towards personalised medicine.