The clinical challenge in the successful management of oral cancer malignancy remains in the inaccuracy of detecting regional invasion potential and inefficient treatment of recurrent tumors. The presence and extent of bone invasion by the oral tumor are of critical importance as they can influence the preoperative strategy altering the prognosis outcome. Here, we are examining the patient-specific osteotropism of oral carcinoma using a bone derived extracellular matrix. The extracellular matrix (ECM) was obtained from caprine bone by a combination of demineralization, delipidation and decellularization (D3) techniques. The bone D3-derived ECM (BdECM) tissue was characterized for analyzing the effective removal of cells, minerals, and lipids with an intact structure and chemical composition. The human adipose-derived stem cells (ADSCs) on the osteomatrix (BdECM derived hydrogel) exhibited excellent cell viability and early osteogenic differentiation capacity in vitro. Furthermore, the osteomatrix polarized monocytes towards an anti-inflammatory phenotype (M2 macrophage) indicating its low immunogenicity. In the second phase of this study, we isolated and established primary cancer cell cultures from patient-derived tissue exhibiting the cancer stem cell marker phenotype (EpCAM+/CD44high/CD24−). Moreover, the presence of side population (SP) cells confirmed a contributing factor for resistance to cancer therapy. The spheroid formed from primary cells embedded in the osteomatrix was used as a test-bed to monitor the invasion profile and screening of anti-cancer drugs. Our 3D test platform captured the inter-patient heterogeneity by displaying variation in the degree of invasion and response towards tested doses of anticancer drugs. Altogether, our data emphasize the necessity of a tissue-specific in vitro preclinical model for the evaluation of oral carcinogenesis and drug sensitivity. © 2023 The Royal Society of Chemistry.