Nanomedicine-based delivery with non-invasive techniques is a promising approach to increase local drug concentration and to reduce systemic side effects. Focused ultrasound (FUS) has become a promising strategy for non-invasive local drug delivery by mild hyperthermia. In this study, traditional temperature-sensitive liposomes (TTSLs) encapsulating doxorubicin (DOX) were evaluated for FUS-mediated drug delivery with an in-vitro FUS setup. In-vitro studies showed quantitative release of the DOX from the lumen of the temperature-sensitive liposomes when heated to 42 °C with FUS using 1 MHz sinusoidal waves at 1.75 MPa for 10 min. No release was observed when heated at 37°C. Moreover, we showed that DOX released from TTSLs by FUS is as efficiently internalized by glioblastoma cells as free DOX at 37°C. In-vitro therapeutic evaluation showed that exposure of a cell monolayer to FUS-activated TTSLs induced a 60% and a 50% decrease in cell viability compared to cell medium and to TTSLs preheated at 37°C, respectively. Using an in-vitro 3D cell culture model, the results showed that after FUS-mediated hyperthermia, preheated liposomes induced a 1.7-fold decrease in U-87 MG spheroid growth in comparison to the preheated liposomes at 37°C. In conclusion, our results show that in-vitro FUS allows the evaluation of TTSLs and does not modify the cellular uptake of the released DOX nor its cytotoxic activity.