The chitosanase-catalyzed hydrolysis of chitosan oligosaccharides was investigated for the first time by real-time electrospray ionization-mass spectrometry (ESI-MS). As chitosan oligosaccharides (GlcNn, n=2-6) were hydrolyzed by exochitosanase (exo-beta-glucosaminidase) from Amycolatopsis orientalis, the reaction time-courses of substrate, intermediate and products could be monitored simultaneously by direct infusion of the reaction solvent into the mass spectrometer. Consequently, the analytical approach of real-time MS is an enormous time-saving method. Furthermore, the high sensitivity of the mass spectrometric detection allows the determination of the reaction time-courses with very low quantities of substrate and therefore also a low amount of applied enzyme. Real-time mass spectrometric detection was also applicable in investigating the reaction behaviour of Streptomyces sp. N174 endochitosanase wild type and of two of its mutants. This technique establishes the fast and efficient determination of in vitro enzymatic activities of various enzyme systems.