Overcoming transport barriers to delivery of therapeutic agents in tumors remains a major challenge. Focused ultrasound (FUS), in combination with modern nanomedicine drug formulations, offers the ability to maximize drug transport to tumor tissue while minimizing toxicity to normal tissue. This potential remains unfulfilled due to the limitations of current approaches in accurately assessing and quantifying how FUS modulates drug transport in solid tumors. We developed a novel acoustofluidic platform by integrating a physiologically relevant 3D microfluidic device and a FUS system with a closed-loop controller to study drug transport and assess the response of cancer cells to chemotherapy in real time using live cell microscopy. FUS-induced heating triggered local release of the chemotherapeutic agent doxorubicin from a liposomal carrier and resulted in higher cellular drug uptake in the FUS focal region. This differential drug uptake induced locally confined DNA damage and glioblastoma tumor cell death in the 3D environment. Our study demonstrates the capabilities of acoustofluidics for accurate control of drug release and monitoring of localized cell response in a 3D in vitro tumor model and has important implications for developing novel strategies to deliver therapeutic agents directly to the tumor tissue while sparing healthy tissue.