Hyaluronan (HA) is a dense compound that is a ubiquitous component of the extracellular matrix in most tissues. However, HA tends to be overexpressed in pancreatic ductal adenocarcinoma (PDAC). This increased HA is associated with high tumor interstitial pressures and microvascular collapse. These conditions collectively compromise microvascular function and impede systemic delivery of anticancer drugs. The aim of this preclinical study was to monitor changes in PDACs following HA degradation by using a real-time multimodal US approach integrating both contrast-enhanced US (CEUS) and photoacoustic (PA) imaging. Athymic nude mice with pancreatic tumors received a single dose of targeted HA or sham treatment. In vivo multimodal US images of tumor tissue were acquired at baseline before dosing and again at 6 h along the same tumor cross-section. Animals were euthanized after imaging at 6 h and tumors were then excised for histological processing. In PDAC-bearing animals, CEUS-derived tumor perfusion measurements were significantly increased at 6 h after dosing with a targeted HA drug as compared to those administrated control treatment (p < 0.04). PA measures of tumor oxygenation were also considerably higher in treated tumors (p = 0.02). Changes in the multimodal US findings were supported by histological analysis of excised tumor tissue samples, which revealed HA depletion (p < 0.001) and increased microvascular patency in the targeted HA treated PDACs. This study reveals PDAC changes following targeted HA treatment can be detected using multimodal US imaging.