Ultrasonography is a powerful imaging modality that enables noninvasive, real-time visualization of abdominal organs and tissues. This technology may be adapted for use in mice through the utilization of higher frequency transducers, allowing for extremely high-resolution imaging of the mouse pancreas. This technique is particularly well suited to pancreas imaging due to the ultrasonographic properties of the normal mouse pancreas, easily accessible imaging planes for the head and tail of the mouse pancreas, and the comparative difficulty in imaging the mouse pancreas with other technologies. A suite of measurement tools is available to characterize the normal and diseased states of tissues. Of particular utility for cancer applications is the ability to use tomography to construct a 3D tumor volume, enabling longitudinal imaging studies to track tumor development, or response to therapies. Here, we describe a detailed method for performing high-resolution ultrasound to detect and measure pancreatic lesions in a genetically engineered mouse model of pancreatic ductal using the VisualSonics Vevo2100 High Resolution Ultrasound System. The method includes preparation of the animal for imaging, 2D and 3D image acquisition, and post-acquisition analysis of tumor volumes. The combined procedure has been utilized extensively by our group and others for the preclinical evaluation of novel therapeutic agents in the treatment of pancreatic ductal adenocarcinoma (Olive et al., Science 324:1457-1461, 2009; Cook et al., Methods Enzymol 439:73-85, 2008; Singh et al., Nat Biotechnol 28:585-593, 2010; Beatty et al., Science 331:1612-1616, 2011).