Microspheres based on the bioactive glass S53P4 were successfully produced through flame-spraying. Glass 13–93 microspheres were utilised as a reference composition with a known behaviour in flame spheroidisation. Bioactive glass microspheres (BGMs) enable a range of advantages compared to conventional irregular-shaped particles. The produced BGMs in size ranges of 45–90 and 90–125 µm were characterised by DTA, SEM/EDXA, FTIR, and XRD. Dynamic dissolution experiments were carried out using TRIS buffer and SBF (pH 7.4) for 72 h. The ion concentrations in the solutions were measured using ICP-OES. The in vitro bioactivity results indicated that the hydroxyapatite (HA) layer was thicker for the larger spheres after dissolution in SBF. According to the SEM-EDXA results, the S53P4 spheres showed a more rapid formation of the hydroxyapatite layer than the reference 13–93. The thermal and in vitro properties of the BGMs were similar to the particles of the parent glasses. The results suggest that microspheres based on S53P4 bioactive glass have an excellent potential for future clinical applications.