We report our approach to creating a microfluidic chip (namely UroLOC) that mimics the acinar/tubular structure and the luminal microenvironment of exocrine glands. The chip utilises a nanostructured membrane that is designed to provide a 3-dimensional supporting scaffold for the growth of exocrine acinus epithelial cells. The nanostructured membrane was produced using layer-by-layer assembly of polyelectrolytes, and formed into 3-dimensional hemispherical cavities and “finger-like” structures in order to mimic the natural architecture of acini found in exocrine glands. We utilised normal (PNT2) and cancerous (PC3, LNCaP) prostate epithelial cells to demonstrate the proof-of-concept of using MALDI (Matrix Assisted Laser Desorption Ionisation) profiling of secretions collected after 48 hours of cell growth, with no concentration or purification steps and without any a priori on the knowledge of targeted proteins. This MALDI profiling analysis of the crude supernatants from 3 different cell lines (PNT2, PC3 and LNCaP) demonstrated the capacity of the MALDI profiling approach to discriminate between the different secretome signatures. The UroLOC concept and secretome profiling that we describe opens new opportunities in terms of liquid-biopsy based diagnosis, particularly for the early stages of carcinogenesis.