The use of novel tools to understand tumour-fibrosis in pancreatic ductal adenocarcinoma (PDAC) and novel anti-fibrotic treatments are highly needed. We established a pseudo-3D in vitro model including humane pancreatic fibroblasts (PFs) and pancreatic cancer-associated fibroblasts (CAFs) in combination with clinical collagen biomarkers, as a translational anti-fibrotic drug screening tool. Furthermore, we investigated the prognostic potential of serum collagen biomarkers in 810 patients with PDAC. PFs and CAFs were cultured in Ficoll-media. Cells were treated w/wo TGF-ß1 and the anti-fibrotic compound ALK5i. Biomarkers measuring the formation of type III (PRO-C3) and VI (PRO-C6) collagens were measured by ELISA in supernatant at days 3, 6, 9, and 12. PRO-C3 and PRO-C6, and their association with overall survival (OS), were evaluated in serum with PDAC (n = 810). PRO-C3 and PRO-C6 were upregulated in CAFs compared to PFs (p < 0.0001.). TGF-ß1 increased PRO-C3 in both PFs and CAFs (p < 0.0001). The anti-fibrotic compound ALK5i inhibited both PRO-C3 and PRO-C6 (p < 0.0001). High serum levels of PRO-C3 and PRO-C6 in patients with PDAC were associated with short OS (PRO-C3:HR= 1.48, 95%CI:1.29–1.71, p < 0.0001 and PRO-C6:HR = 1.31, 95%CI:1.14–1.50, p = 0.0002). PRO-C3 and PRO-C6 have the potential to be used both pre-clinically and clinically as a measure of tumor fibrosis and CAF activity.