Genetically engineered mouse model(GEMM) that develops pancreatic ductal adenocarcinoma (PDAC) offers an experimental system to advance our understanding of radiotherapy (RT) for pancreatic cancer. Cone beam CT (CBCT)-guided small animal radiation research platform (SARRP) has been developed to mimic the RT used for human. However, we recognized that CBCT is inadequate to localize the PDAC growing in low image contrast environment. We innovated bioluminescence tomography (BLT) to guide SARRP irradiation for in vivo PDAC. Before working on the complex PDAC-GEMM, we first validated our BLT target localization using subcutaneous and orthotopic pancreatic tumor models. Our BLT process involves the animal transport between the BLT system and SARRP. We inserted a titanium wire into the orthotopic tumor as the fiducial marker to track the tumor location and to validate the BLT reconstruction accuracy. Our data shows that with careful animal handling, minimum disturbance for target position was introduced during our BLT imaging procedure(<0.5mm). However, from longitudinal 2D bioluminescence image (BLI) study, the day-to-day location variation for an abdominal tumor can be significant. We also showed that the 2D BLI in single projection setting cannot accurately capture the abdominal tumor location. It renders that 3D BLT with multipleprojection is needed to quantify the tumor volume and location for precise radiation research. Our initial results show the BLT can retrieve the location at 2mm accuracy for both tumor models, and the tumor volume can be delineated within 25% accuracy. The study for the subcutaneous and orthotopic models will provide us valuable knowledge for BLTguided PDAC-GEMM radiation research.