The static analysis of tumor tissues at a single, terminal end point has been the mainstay of studies in mouse models of cancer. However, with the development of model systems that reproduce the intra- and intertumoral heterogeneity of human tumors, such studies are limited by the need for large numbers of animals to overcome increased intragroup variance. The shortcomings of a single-timepoint approach to molecular analysis are especially apparent in the context of therapeutic studies, in which the dynamic response to treatment is of particular consequence. To mitigate the effects of intertumoral heterogeneity, multiple tissue samples may be harvested from the same tumor at different time points through the use of surgical biopsies. For abdominal tumors, preprocedure imaging may be used to assess the suitability of tumors for biopsy. Sterile surgical techniques are used to access the abdominal cavity, and customized instruments facilitate the immobilization and retrieval of tissue samples from hard or fibrous tumors. Thermoregulation, hemostasis, and wound closure techniques are critical to successful surgical outcomes, whereas appropriate anesthetic, analgesic, and postoperative recovery regimens are important for maintenance of animal welfare. Using a mouse model of pancreatic ductal adenocarcinoma, we present a comprehensive protocol suitable for the routine acquisition of abdominal tumor biopsies.