This project investigated cell-derived extracellular matrix (ECM) preparation for matrix-guided bone tissue engineering. A detergent-based decellularisation protocol using NH4OH and Triton X-100 was optimised using fibroblast-derived ECM to improve DNA removal while preserving ECM-associated protein content. To provide a more bone-relevant molecular reference, the same decellularisation approach was also applied to HOB-derived ECM. Proteomic analysis of HOB-derived dECM identified ECM-associated proteins linked to osteogenic activity, including collagens, laminins, annexins, and calcium-binding proteins. This dataset is presented as a descriptive reference for osteoblast-derived ECM composition, rather than as a direct comparison with the fibroblast-derived ECM used in functional assays.