The ex vivo modelling of pancreatic ductal adenocarcinoma (PDAC) using patient-derived cells is a promising tool to predict treatment responses. Matrigel-based organoid and organotypic approaches are limited by their undefined molecular composition, hindering the recapitulation of the tumour’s characteristic desmoplasia, which is known to promote drug resistance. To overcome these limitations, we used self-assembling peptide amphiphiles (PAs) gelled in a minimal extracellular matrix to model the pancreatic tumour microenvironment and to establish 3D multicellular cultures of patient-derived PDAC cells, pancreatic stellate cells and macrophages. Matrisome analysis of 3D cultures demonstrated consistent ECM protein deposition, which was highly reminiscent of the corresponding primary PDAC tissues. The proteomic data obtained was also compared to the corresponding patient-derived xenografts (PDX) in nude mice and Matrigel-based cultures. Characterisation of the chemosensitivity of the cultures revealed realistic treatment responses by PDAC cells in PA hydrogels based on the responses of their corresponding PDX tumours. Histological, transcriptional and functional techniques confirmed these similarities, which were not observed in Matrigel-based cultures. These findings demonstrate the biomimetic nature of PA hydrogels, which enable cultured cells to recreate the PDAC matrisome ex vivo and to respond to chemotherapeutic agents in a predictive manner.