A number of preclinical and clinical studies with chitosan-adjuvanted antigen- and DNA-based vaccines have been carried out. Various chitosans and their modifications, in different forms (solutions, powders, gels and particles), have been evaluated with various antigens administered via different routes. Chitosan is a generic name for a wide array of glucosamine-based substances derived from biological sources, and standardization is necessary. However, in most of the studies published to date, molecular weight, viscosity, deacetylation degree and/or purity level (especially endotoxins) are not provided for the initial chitosan substance and/or final formulation and the preparation procedure is not detailed. Evaluation of adjuvant properties is challenging, given that the only available data are insufficient to demonstrate immunogenicity for chitosans with characteristics within certain intervals to elucidate mechanisms of action or to exclude impurities as the active substance. These and other issues of chitosan-based vaccine adjuvants are summarized and a step-by-step evaluation approach for chitosan-based vaccine adjuvants is outlined.