Interactions between neoplastic epithelial cells and components of a reactive stroma in pancreatic ductal adenocarcinoma (PDAC) are of key significance behind the disease's dismal prognosis. Despite extensive published research in the importance of stroma-cancer interactions in other cancers and experimental evidence supporting the importance of the microenvironment in PDAC progression, a reproducible three-dimensional (3D) in vitro model for exploring stroma-cancer interplay and evaluating therapeutics in a physiologically relevant context has been lacking. We introduce a humanized microfluidic model of the PDAC microenvironment incorporating multicellularity, extracellular matrix (ECM) components, and a spatially defined 3D microarchitecture. Pancreatic stellate cells (PSCs) isolated from clinically-evaluated human tissue specimens were co-cultured with pancreatic ductal adenocarcinoma cells as an accessible 3D construct that maintained important tissue features and disease behavior. Multiphoton excitation (MPE) and Second Harmonic Generation (SHG) imaging techniques were utilized to image the intrinsic signal of stromal collagen in human pancreatic tissues and live cell-collagen interactions within the optically-accessible microfluidic tissue model. We further evaluated the dose-response of the model with the anticancer agent paclitaxel. This bioengineered model of the PDAC stroma-cancer microenvironment provides a complementary platform to elucidate the complex stroma-cancer interrelationship and to evaluate the efficacy of potential therapeutics in a humanized system that closely recapitulates key PDAC microenvironment characteristics.