In order to grow, infiltrate and spread throughout the body, heterogeneous tumour systems often utilise proteolytic and fibrinolytic pathways, through activation of the plasminogen activation pathway. Receptor bound urokinase (uPA) is the main activator of this pathway and has been identified as a major cell marker of tumour invasion and metastasis, proven as a significant therapeutic target. The naturally occurring inhibitor of uPA, plasminogen activator inhibitor type-2 (PAI-2/serpinB2), specifically and irreversibly binds to uPA/uPAR and the complex is internalised into the cell via receptor-mediated endocytosis. This interaction prevents plasminogen from conversion into plasmin, which otherwise results in degradation of stroma, and activation of an extensive reservoir of growth factors (VEGF, bFGF, TGF-β), the matrix metalloproteinases, many downstream kinase pathways (ERK1, MAPK), ultimately commanding advanced proliferation, apoptotic suppression, invasion and metastasis, within a tumourigenic environment. To define the tumour versus host specific effects of PAI-2, and the role of its serpin and/or other potential functions in the metastatic processes, we are using organotypic assays, which utilise fibroblast-contracted collagen 1 dermal equivalent matrices, as malleable platforms for the assessment of tumour/stromal interactions in a 3D context. Here, we report the effect of overexpressing or silencing PAI-2 expression on MDA-MB-231HM breast and Kras/Trp53 mutated pancreatic ductal (PDAC) adenocarcinoma tumour cell invasion in this assay. The effect of modifying fibroblast PAI-2 expression was also tested in this 3D system in order to elucidate the variant PAI-2 expression in tumour cell versus stroma upon invasion. Furthermore, this assay can be used for drug assessment prior to in vivo testing. We found wild-type PAI-2 to significantly inhibit the invasion of uPA-overexpressing PDACs compared to controls (p < 0.0001). An inactive PAI-2 mutant, R380APAI-2, had no significant effect on PDAC invasion confirming uPA-dependent inhibition of invasion (p > 0.05). Therefore, this study further supports biological rationale for the continued development of anti-uPA targeted therapies.