Since the first report of mesoporous silica nanoparticles in 2001, many efforts have been made to develop them for biomedical applications. With the emergence of new designs and increasingly complex synthetic schemes, mesoporous silica nanoparticles have never been more promising. For this promise to be fulfilled, however, practical considerations for biomedical use must be carefully addressed. Many current mesoporous silica reports, even those reporting in vivo work, neglect the observation of nanoparticle size, pore structure, aggregation state, and biodegradability under biological conditions before administration. These critical considerations, beginning at synthetic stages, must be taken into account to make effective and safe mesoporous silica nanoparticles for biomedical use and timely application in clinical trials. Herein, we present a comprehensive review of mesoporous silica nanoparticle synthetic strategies, pointing out nanoparticle behavior under biological conditions and how it may affect in vitro and in vivo results.