Static and dynamic in vitro dissolution studies showed large differences for various size-fractions of non-porous, flame-sprayed commercial microspheres (45–500 µm) of bioactive glass S53P4. The smaller the spheres, the more their composition deviated from the nominal glass. The dissolution studies were carried out in simulated body fluid and tris(hydroxymethyl) aminomethane buffer for seven days. The ion concentrations in solutions were analyzed using inductively coupled plasma optical emission spectrometry, and the pH was measured as a function of time. Also, changes in the sphere size distribution and mass losses were determined. The calcium phosphate and the silica-rich layers at the sphere surfaces were investigated using scanning electron microscopy after several immersion times. The smallest (45–90 µm) spheres appeared almost inert. In contrast, typical silica-rich and calcium phosphate layers were identified at the largest spheres after three days of static and dynamic dissolutions. During the past years, bioactive glass microspheres have been added to paste-like injectable bone grafting materials, putties to enhance their molding properties. The obtained results provide a better understanding of the dissolution patterns of bioactive glass microspheres.