Background: Heterotopic patient-derived xenografts (PDX) have been used to assess response to therapy however they underrepresent the role of tumor microenvironment and rarely develop metastasis, both of which are overcome by orthotopic models. Fluorescent orthotopic mouse models require invasive measures to determine tumor bioluminescence. Ultrasonography (US) is a cost-effective, non-invasive imaging technique that has been used in genetically engineered mouse models of pancreatic cancer for a three-dimensional (3D) acquisition of tumor volume, which allows rapid and safe in vivo drug testing. We intend to demonstrate that this technique allows real time monitoring of in vivo response to therapy using patient-derived orthotopic xenograft (PDOX) of pancreatic ductal adenocarcinoma (PDAC). Methods: A non-survival study using PDOX was designed with control (n = 5) and treatment (n = 5) groups. Weekly 3D US images were obtained pre and post-treatment over 4 weeks. Tumor growth curves were generated to monitor progression of disease. Metastatic burden was determined during necropsy. Results: One mouse was excluded from control and treatment groups due to baseline tumor size exceeding 300mm³ and drug toxicity, respectively. Pre-treatment average tumor volumes for control and treatment groups were (36±12)mm³ and (34±12)mm³, respectively. No difference was found in average tumor growth over time between groups (p = 0.9120). A 20% tumor regression was observed per group. Both groups exhibited gross metastasis to spleen, peritoneum, and omentum. Liver, periportal metastasis and local extension to the gastrointestinal and genitourinary system were present on the treatment group. Conclusions: This study describes a rapid technique for in vivo drug response by using 3D US to monitor PDOX; failure of response to therapy correlated with metastatic burden observed. PDOX regression could be explained by tumor heterogeneity. PDOX models, as challenging as they could be, remain to be necessary in vivo models to show therapeutic response to human PDAC, which could be easily monitored using 3D US imaging.