Both sulfated hyaluronic acid and dexamethasone are candidates for the functionalization of bone grafts as they were shown to enhance the differentiation of osteoblasts from bone marrow stromal cells in vitro and in vivo. However, the underlying mechanisms are not fully understood. Furthermore, studies combining different approaches to assess to synergistic potentials are rare. In this study, we aim gain insights into the mode-of-action of both sulfated hyaluronic acid and dexamethasone by a comprehensive analysis of the cellular fraction, released matrix vesicles and the extracellular matrix, combining classical biochemical assays with mass spectrometry-based proteomics and further supported by novel bioinformatical computation.