High-intensity focused ultrasound (HIFU) is a promising means of non-invasive therapy for the treatment of tumors. Monitoring and assessment challenges for HIFU therapy remain, however, and ultrasound is under investigation to accomplish non-invasive treatment feedback. Quantitative ultrasound (QUS) was investigated for acute HIFU therapy assessment as well as temperature monitoring in a rat model mammary tumor both in vivo and ex vivo. In the in vivo study, 40 rodent mammary tumors were exposed to focused ultrasound produced by a 1-MHz single-element transducer (f/1.1) at three spatial-peak temporal-average intensity levels (335, 360, and 502 W/cm2). Ultrasound assessment scans were performed on each tumor before and again immediately after HIFU exposure using clinical (Ultrasonix L14/5, 3-8 MHz) and small-animal high-frequency (VisualSonics MS-200, 6-14 MHz) ultrasound systems.