Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest cancer types and is estimated to cause more than 432,242 death per year in 2018. Despite increased knowledge in cancer biology and advances in diagnosis tools, most patients were diagnosed at the malignant stage, unsuitable for surgical resection. Moreover, current treatment options like chemotherapy and radiation therapy have limited effect at the malignant stage and only serve as palliative care. Recently, immunotherapy like CAR-T cell therapy has gained great success in some cancer types. To perform the antitumor activities, T cells need to recognize tumor antigens by either establishing contacts with cancerous cells or antigen-presenting cells, which depends on a series of migratory steps from entry to the tumor to the locating of the malignant cells. The dense stroma associated with PDAC creates a great challenge for T cells to effectively migrate in the tumor microenvironment (TME). Also, the mechanism for T cell navigation and migration is not well understood. Here we presented a rapid and cost-effective method to fabricate 3D collagen gel matrix that closely assembles the remodeled extracellular matrix (ECM) in PDAC, allowing high throughput studies. To fully recapitulate the naïve TME, tumor slices from the KPCT genetically engineering PDAC mouse model that shares many of the disease characteristics of human PDAC can be prepared and cultured in an organotypic culture insert for up to six days while maintaining tissue integrity and cell viability. CD8⁺+ can be isolated from tumorbearing KPC/KPCT mice and activated using Dynabeads T cell activator. Fully activated CD8⁺+ can then be seeded on top of the tumor slice, and attached/infiltrated T cell migration can be monitored using time-lapse imaging utilizing multiphoton laser scanning microscope (MPLSP), and tumor collagen stroma can be virtualized using second harmonic generation (SHG). Additionally, Trackmate was used to track the cytotoxic T lymphocyte migrations in the TME. The 3D speed and motility of the T cells were further analyzed in carcinoma cell-rich and stroma-rich regions of the KPCT TME. Our data suggested that T cell speed and motility both increased significantly in the carcinoma-rich region, where collagen fibers are loosely connected, than that of the stroma-rich region. Taken together, the live imaging approach combined with quantitative analysis allows us to gain insight into T cell migration in the complex 3D TME and form new therapeutic approaches.