Among several commercial enzymes screened for chitosanolytic activity, Neutrase 0.8L (a protease from Bacillus amyloliquefaciens) was selected in order to obtain a product enriched in deacetylated chitooligosaccharides (COS). The hydrolysis of different chitosans with this enzyme was followed by size exclusion chromatography (SEC-ELSD), mass spectrometry (ESI-Q-TOF), and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Neutrase 0.8L converted 10 g/L of various chitosans into mostly deacetylated oligosaccharides, yielding approximately 2.5 g/L of chitobiose, 4.5 g/L of chitotriose and 3 g/L of chitotetraose. We found out that the neutral protease was not responsible of the chitosanolytic activity in the extract, whilst it could participate in the deacetylating process. The synthesized COS were tested in vitro for their neuroprotective (towards human SH-S5Y5 neurons) and anti-inflammatory (in RAW macrophages) activities, and compared with other functional ingredients, namely fructooligosaccharides.