Description: Background: There is a strong need for early assessment of tumor response to chemotherapy in order to avoid the adverse effects of unnecessary chemotherapy and to allow early transition to second-line therapy. The purpose of this study was to determine the feasibility of ultrasonic spectral analysis for the in vivo characterization of changes in tumor microstructure in the evaluation of tumor response to chemotherapy using diagnostic ultrasound. Methods: Experiments were approved by the regional animal care committee. Twenty-four MCF-7 breast cancer bearing nude mice were treated with adriamycin or sterile saline administered by intraperitoneal injection. Ultrasonic radio-frequency (RF) data was collected using a clinically available ultrasound scanner (6-MHz linear transducer). Linear regression parameters (spectral slope and midband-fit) regarding the calibrated power spectra from the RF signals were tested to monitor tumor response to treatment. The section equivalent to the ultrasound imaging plane was stained with hematoxylin and eosin to allow for assessment of the density of tumor cell nuclei. Results: Treatment with adriamycin significantly reduced tumor growth in comparison with the control group (p = 0.003). Significant changes were observed in the ultrasonic parameters of the treated relative to the untreated tumors (p 〈 0.05). The spectral slope increased by 48.5%, from -10.66 +/- 2.96 to -5.49 +/- 2.69; the midband-fit increased by 12.8%, from -57.10 +/- 7.68 to -49.81 +/- 5.40. Treated tumors were associated with a significant decrease in the density of tumor cell nuclei as compared with control tumors (p 〈 0.001). Conclusions: Ultrasonic spectral analysis can detect changes in tumor microstructure after chemotherapy, and this will be helpful in the early evaluation tumor response to chemotherapy.