Irinotecan is a powerful anticancer drug with severe systemic side effects that limit its clinical application. Drug-targeted delivery with noninvasive methods is required to enhance the drug concentration locally and to reduce these undesirable events. Microbubble-assisted ultrasound has become a promising method for noninvasive targeted drug delivery. The aim of this study is to evaluate the therapeutic effectiveness of in vitro and in vivo irinotecan delivery based on the combination of ultrasound and microbubbles. In the present study, in vitro results showed that the irinotecan treatment with microbubble-assisted ultrasound induced a significant decrease in cell viability of human glioblastoma cells. Moreover, using subcutaneous glioblastoma xenografts, the in vivo preclinical study in nude mice demonstrated that this therapeutic protocol led to a decrease in tumor growth and perfusion and an increase of tumor necrosis. The conclusions drawn from this study demonstrate the promising potential of this therapeutic approach for the anticancer targeted therapy.