Partially acetylated chitosan oligosaccharides (paCOS), consisting of β-1,4-linked N-acetyl-d-glucosamine and d-glucosamine units, possess diverse bioactivities that can be used for applications in, e.g., biomedicine, agriculture, and pharmaceutics. Establishing structure-function relationships and revealing modes of action requires the availability of structurally defined paCOS that can best be produced using chitin- and chitosan-modifying enzymes, such as chitinases and chitosanases, with known and defined subsite specificities. To enlarge the spectrum of such enzymes and, consequently, defined paCOS available, we have developed a two-step, microtiter plate-based high-throughput screening assay that allows quantification of the activity and subsite specificities of chitosan hydrolases. In a first step, the activities of the enzymes are quantified using a reducing end assay, and enzymes with sufficient activity are then screened for their subsite specificities using mass spectrometric analysis of their products when acting on well-defined chitosan polymers as substrates. The rapid UHPLC-ELSD-ESI-MS2 method does not require labeling steps or addition of standards, and the principal component analysis of the fragment ion intensities of just two isomeric oligomer groups, GlcNAc1GlcN3 and GlcNAc2GlcN2, sufficed to identify, in a directed evolution, the site-saturation mutagenesis library of Bacillus sp. MN chitosanase consisting of 167 muteins, enzymes that significantly differed in their subsite specificities from the wildtype enzyme. Detailed analyses of a few selected muteins proved that the screening method is efficient and accurate in predicting altered subsite specificities.