Abstract. Hyperosmotic agents have shown great potential in tissue optical clearing. However, the low efficiency of the permeation in biological tissues seriously restricts its application in reality. The synergy of sonophoresis as a penetration enhancer and hyperosmotic agents, 20% glucose (G) and 20% mannitol (M), in optical clearing has been investigated by analyzing the variation of the attenuation coefficients and the permeability coefficients. In the sonophoresis experiments, ultrasound (US) was applied for 10 min before applying hyperosmotic agents. Along with the administration of hyperosmotic agents, the samples were monitored with optical coherence tomography (OCT) functional imaging for the next 2 h. The attenuation coefficients of each group were obtained from the 2-D OCT images based on Beer's Law. The original attenuation coefficient is 12.38 AE 0.73 cm −1 in normal breast tissue. After 45 min treatment, it changes to be 5.91 AE 0.82 cm −1 and 4.14 AE 0.67 cm −1 for 20% G and 20% G/US, respectively. The attenuation coefficient of breast cancer tissue is 18.17 AE 1.45 cm −1 at the beginning, and it becomes 8.70 AE 0.87 cm −1 for 20% G and 6.80 AE 0.92 cm −1 for 20% G/US after 30 min. Meanwhile, the permeability coefficients of hyperosmotic agents were much enlarged by the treatment of ultrasound in both breast normal tissue and breast cancer tissue. A significant difference in permeability coefficients between health tissue and tumor tissue was also observed in the experiment (p < 0.01).