Because of their excellent degradability and bioactivity in the human body, bioactive glasses of wide compositional range are synthesized for regenerative medicine and therapeutic functions. In this paper, the bioactivity of SiO2(45)CaO(25)Na2O(30-x)P2O5(x) x = 0,1,3,5 glasses was tested in vitro by soaking for 30 min in simulated body fluid. Bioactive surface layer formation after this short treatment was assessed by focused ion beam assisted scanning electron microscopy and nanoindentation. Based on the nanoindentation tests, a novel method for quantitative assessment of bioactive layer formation is presented. Connection between the mechanical properties and glass network structure parameters is established up to the theoretical yield strength. It is demonstrated that the ratio of hardness and Young's modulus provides estimation of the structural evolution during simulated body fluid treatments and thus, in the initial stage of bioactive layer formation, serves as a good parameter for measuring the bioactivity.