Pancreatic cancer, particularly pancreatic ductal adenocarcinoma (PDAC), is a highly fatal malignancy and the tumor microenvironment plays an important role in its tumorigenesis. The improvement of treatment methods has been stagnant during the last decade, which has engendered the need for new and enhanced treatments. Spheroids are clusters of cancer cell colonies within a solid tumor, including PDAC. Establishment of an in vitro model of PDAC spheroid formation would provide great potential for studying the biological properties of tumors as well as for drug screening and therapeutic efficacy evaluation. However, current methods of generating spheroids usually take from weeks to months, which is inefficient, particularly to determine which, if any, chemotherapy drugs will cause a response for PDAC personalized therapy. Thus, the aim of this Master thesis project was to establish an effective method for generating PDAC spheroids in 3D culture. Six series of experiments in 2D and 3D cultures were performed with different techniques and protocols. The cell line of UN-KC-6141, originally derived from transgenic mouse model of PDAC was used for this purpose. Modifications and optimizations of culture environment, particularly with a mixture of peptides (so-called DBM peptides) were performed. Detections of cell proliferation and morphology at light and electron microscopical levels were performed. Proteomics and bioinformatics, using Ingenuity Pathway Analysis (IPA) were applied to study the underlying molecular mechanisms of spheroid formation. Formation of spheroids in high quantity and quality was achieved within 3 days in 3D culture in comparison to 52 days in 2D culture. The spheroids displayed one dense core per spheroid. 861 specific peptides in the culture environment were identified, and signaling pathways, such as mTOR, EIF2, ILK, clathrin-mediated endocytosis and caveolar-mediated endocytosis, MAPK, MEK1, ERK and MLCK, were involved in the peptides-enhanced spheroid formation. In conclusion, an effective method of generating spheroids in 3D culture was established using the hanging drop method with peptides-optimized culture environment. This method could be used in the future not only for evaluating drug treatments and screening new drugs, but also for studying tumor biology, tumorigenesis, and morphogenesis of pancreatic cancer.