Three novel borosilicate bioactive glasses (BBGs) of general formula of 0.05Na2O·0.35x·0.20B2O3·0.40SiO2 (molar ratio, where x = MgO or CaO or SrO) were prepared and used to investigate the effect of crystallization on their properties including cytotoxicity. The three postmelt compositions were determined using X-ray fluorescence spectroscopy, and crystallization events were studied using differential thermal analysis and X-ray diffraction. This information was used to determine heat treatments to prepare glass-ceramics by controlled crystallization. X-ray diffraction analysis and Fourier transform infrared spectroscopy showed that, after higher heat treatment temperatures (800–900 °C), borosilicate bioactive glass-ceramics (BBGCs) contained mainly borate and silicate crystalline phases. Specifically, BBG-Mg, BBG-Ca, and BBG-Sr glass-ceramics detected the presence of magnesium silicate-Mg2(SiO3)2 and magnesium borate-Mg2B2O5; wollastonite-2M-CaSiO3 and calcium borate-Ca(BO2)2; and strontium silicate-SrSi...