The purpose of this study was to monitor acute changes in pancreatic tumor perfusion with contrast-enhanced ultrasound (CEUS) imaging following targeted hyaluronan (HA) treatment. Intratumoral accumulation of HA is one of contributing factors that can lead to an increased tumor interstitial pressure (TIP). These elevated TIP levels can hinder delivery of chemotherapeutic drugs and cause treatment failure. For this study, pancreatic cancer-bearing mice were imaged at baseline and again at 2 h after intravenous administration of physiological saline (control group) or PEGPH20, which targets HA (therapy group). CEUS data were collected for 5 min and the temporal sequence was first analyzed using a singular value filter (SVF) to remove any background clutter signal. Given the time history of contrast agent flow, a tumor perfusion parametric analysis was performed. A series of morphological image operations was applied to quantify structural features of the tumor angiogenic network including vessel count, density, length, diameter, tortuosity, and branching points. After imaging, animals were euthanized, and tumors excised for histological processing. Acute microvascular changes were found at 2 h after drug administration as confirmed by CEUS imaging. Further, histologic analysis of tumor sections revealed lower HA accumulation in the therapy group animals. Overall, these findings suggest that CEUS imaging of acute changes in tumor perfusion may help identify an optimal window whereby follow-up chemotherapeutic drug dosing would be more effective.