Extracellular vesicles (EVs) are phospholipid bilayer-enclosed nanoparticles secreted by most cells into the biofluids. They have an important role in intercellular communication by carrying bioactive components (e.g., lipids, proteins, nucleic acids, and other metabolites) and reflecting the biochemical and metabolic processes of their parent cell. Due to their widespread molecular cargo, they have potential diagnostic and therapeutic roles, so they can be considered as new generation biomarkers. With the drastic increase in the number of publications and the comparability of the obtained results, the development of standardized protocols has become necessary. Here we aim to demonstrate the applicability of two, non-conventional techniques, such as ATR-infrared spectroscopy (ATR-FTIR) and electromagnetic piezoelectric acoustic sensor (EMPAS) in the characterization of compositional changes of blood- and pancreatic tumor-derived vesicles. In addition, we provide guidelines in sample collection, isolation, and separation of EVs. IR spectroscopy, as a fast and label-free technique, gives biochemical insight into the sample composition, while EMPAS serves information on vesicle quantity and in membrane integrity changes during storage.