Microbubble-mediated ultrasound therapy has been shown to noninvasively increase drug uptake through increasing cell membrane permeability and vascular extravasation. This technique has potential to improve drug uptake in localized areas of cancer. Using optical imaging to monitor fluorescent tracer uptake is a non-invasive method for increasing information on microbubble-mediated ultrasound therapy. Mice (N = 10) were implanted with 2LMP breast cancer cells and injected with Cy5.5 fluorescent dye before undergoing microbubble-mediated ultrasound therapy. Post therapy, animals were transiently imaged at 1, 10, 30 and 60 min using a small animal optical imaging system. Tumors were extracted and analyzed for fluorescent signal. Using ROI analysis, the microbubble-mediated ultrasound therapy group showed increased uptake compared to control group (p = 0.02). Supernatant from tumors, post extraction, also showed an increased fluorescent dye compared to control counterparts (p = 0.01). In vivo fluorescent imaging of microbubble-mediated ultrasound therapy allows monitoring of uptake in a localized area.