Gelatin microspheres (GMs) using various concentrations of epoxypropoxypropyl-trimethoxysilane (EPPTMS) as a crosslinking agent were prepared. Since the crosslinking agent content and manufacturing conditions are crucial factors in the characteristics of the microspheres, this study was carried out to evaluate the effect of processing variables on morphology, and in vitro behavior of GMs using acellular simulated body fluid (SBF). It was found that the best morphology was acquired in terms of both shape and uniformity for the GMs containing a certain concentration of EPPTMS (20wt%), whereas other concentrations yielded rambling spheres. The results revealed that the size of microspheres became smaller when both the oil-to-water ratio and agitation rate were increased. Moreover, the density of the crosslinking agent was also dependent on the above-mentioned factors in 20wt% EPPTMS. Different morphologies of hydroxyapatite deposits on the surface of GMs were observed after 14 days of soaking in SBF. These in vitro acellular studies were confirmed by SEM, FTIR, and XRD analyses. According to the results of UV-Vis spectroscopy, the GMs were released into the SBF solution in a time-dependent manner; however, the fastest rate of release occurred for the largest GM.