The chorioallantoic membrane (CAM) model resembles a preclinical in vivo platform for the engraftment of different tumors and is especially well-suited for the development and evaluation of novel cancer therapies. Due to the CAM′s extensive vascular network, it can sustain engrafted human tumor tissue, which can be used for therapy testing and analysis throughout the experimental process. Although numerous studies have demonstrated successful tumor engraftment and vascular integration into the CAM′s blood vessel network, the accurate and sequential measurement of tumor volume remains a significant challenge. Current imaging modalities, such as positron emission tomography/computed tomography, magnetic resonance imaging or three-dimensional (3D) digital microscopy are limited by cost, complexity, and potential harm due to injected agents or radiation. This study introduces an innovative imaging approach by utilizing ultrahigh frequency (UHF) ultrasound combined with 3D ultrasound reconstruction of pancreatic ductal adenocarcinoma (PDAC) on the CAM. By utilizing ultrasound frequencies of up to 71 MHz, two-dimensional (2D) imaging of PDAC tumors was transformed into comprehensive 3D reconstructions. This innovative approach provides a more detailed tissue resolution and enables noninvasive, longitudinal volumetric monitoring of tumor growth in vivo, and in principle provides a platform for future studies assessing angiogenesis and therapy response. In this study the accuracy of 3D UHF ultrasound was tested by comparing it with established imaging techniques such as 3D digital microscopy. In summary, the 3D ultrasound reconstruction resembles a more versatile noninvasive alternative for in vivo tumor volume measurement. This advanced imaging approach adds a valuable methodology for the investigation of tumor biology in the CAM model by providing noninvasive volumetric and morphological characterization of engrafted tumor tissue, thereby linking preclinical research and clinical applications.