Vascular abnormalities in a complex tumor microenvironment is one of the major challenges for effective drug delivery and cancer treatment. It has been well established that noninvasive focused ultrasound (FUS) combined with a microbubble (MB) contrast agent can safely and reversibly increase the permeability of blood vessel walls, thereby temporarily allowing anticancer agents to pass through them and into the tumor tissue. Termed FUS-mediated drug delivery, most previous studies have used a single focused US beam for cancer treatment in small animal models, which limited the ability to treat the entire tumor burden. To that end, the goal of this research was to reveal the effectiveness of tumor treatment when using multi-FUS system and method. An infrared dye (IR-780) functioned as a surrogate chemotherapeutic drug and allowed detection in live animals. Athymic nude female mice implanted with MDA-MB-231 breast cancer cells were used to evaluate both single FUS and multi-FUS therapeutic strategies. Each animal was injected in the tail vein with a bolus mixture of MB (2.3 x 107 Definity, Lantheus Medical Imaging) and IR-780 dye (50 μg). During US therapy, a custom pulsed sequence was applied using an image-guided FUS system (HIFUPlex-06, Verasonics Inc) for a duration of 10 min in the selected zone of the tumor. Animals were imaged using a whole-body optical imaging system (Pearl Trilogy, LI-COR Biosciences) and accumulated IR-780 dye was quantified up to 48 h after application of FUS-mediated drug delivery. After euthanasia, IR-780 dye was also quantified from homogenized tumor tissue samples. Overall, preliminary results showed that the multi-FUS therapy approach significantly increased drug uptake (increased by about 71 % at 48 h) in the targeted tumor tissue compared to the single FUS method.