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In this study, the SiO 2 – CaO (SC) and SiO 2 – CaO – P 2 O 5 (SCP) glass powders doped with 0.3 mol% of Tm3+ and 4 mol% of Yb3+ ions were prepared by the sol–gel method. The samples were immersed for four weeks in a simulated physiological environment (Dulbecco's phosphate-buffered saline, DPBS) to investigate the bioactivity of the glasses. Their structure, morphology, and spectroscopic properties were investigated in the function of immersion time. X-ray diffractograms and infrared spectra showed that after incubation in DPBS, the hydroxyapatite starts to build up after 1–3 days. The energy dispersive X-ray spectroscopy analysis indicated the dynamics of ion exchange resulting from the biomineralization process in the glasses. Moreover, the upconversion emission spectra of Tm3+ were measured for all samples under 980 nm excitation wavelength in resonance with the 2F 7/2 → 2F 5/2 absorption of Yb3+. It was observed that the upconversion luminescence continuously decreased and the decay times shorten slightly after the immersion in the buffer. In vitro cytotoxicity assays revealed that the SC glass was biocompatible for all tested cell lines (human chondrocytes, osteoblasts, and fibrosarcoma). However, the SCP powder showed an inhibitory effect on human chondrocyte and fibrosarcoma cells in small concentrations and fibrosarcoma cells in high concentrations. [ABSTRACT FROM AUTHOR]