The increasing knowledge of cellular and molecular mechanisms of human diseases allows envisaging the gene therapy by sonoporation as an emerging and promising therapeutic alternative. Sonoporation combines the local application of ultrasound waves and the intravascular or intratissue administration of gas microbubbles. In such a way, the permeability of vessels and tissues to the poorly permeant molecules is transiently increased. Ultrasound based modality offers new opportunities since ultrasound can be easily focused on a target tissue or organ and hence gene delivery and expression should be limited to the insonified region. Consequently, it might be possible to develop an efficient and safe tissue- or organ-specific delivery method by microbubble targeting and focused ultrasound. This review focuses on the current knowledge of sonoporation fundamentals and mechanisms. The sonoporation procedure and current preclinical trials will be then presented. Finally, the new challenges of sonoporation will be discussed.