Ultrasound-mediated gene transfection (sonotransfection ) has been shown to be a promising physical method for gene therapy, especially for cancer gene therapy. The procedure being done in vitro uses several ultrasound exposure (sonication) setups. Although high transfection rates have been attained in some of these setups in vitro, replicating similar levels of transfection in vivo has been difficult. In vivo-simulated setups offer hope for a more consistent outcome in vivo. Presented in this chapter are typical methods of sonotransfection in vitro, methods when using a novel in vivo-simulated in vitro sonication setup and also sonotransfection methods when doing in vivo experiments. Factors that could potentially influence the outcome of an ultrasound experiment are cited. Several advantages of sonotransfection are recognized, although a low transfection rate is still considered a disadvantage of this method. To improve the transfection rate and the efficiency of sonotransfection, several studies are currently being undertaken. Particularly promising are studies using engineered microbubbles to carry the therapeutic genes into a particular target tissue in the body, then using ultrasound to release or deliver the genes directly into target cells, e.g., cancer cells.