Graphene and graphene oxide can promote the adhesion, growth and differentiation of mesenchymal stem cells. Further, graphene surface coatings accelerate the differentiation of human mesenchymal stem cells acting as osteogenic inducers. Quantification of the osteogenic induction is conventionally performed with Alizarin Red S (ARS), an anthraquinone derivative used to identify calcium deposits in tissue sections and cell cultures. The ARS staining is quite versatile because the dye forms an Alizarin Red S⁻calcium complex that can be extracted from the stained monolayer of cells and readily assayed by absorbance measurements. Direct visualization of stained deposits is also feasible; however, an in-situ visualization and quantification of deposits is possible only on transparent supports and not on thick opaque materials like ceramics and graphene composites that are well-known inducers of osteogenesis. In this manuscript, the shape of the 2D-fluorescence spectra of the ARS-calcium complex is used to develop a method to detect and monitor the in-situ differentiation process occurring during the osteogenic induction mediated by opaque graphene oxide surfaces.