The cationic biopolymer chitosan (CH) has emerged as a promising candidate adjuvant due to its safety profile and immunostimulatory properties. The presence of endotoxin contamination in biomaterials is generally underappreciated and can generate misleading results. It is important to establish a convenient methodology to obtain large amounts of high quality chitosan nanoparticles for biomedical applications.We developed an easy method to generate endotoxin-free chitosan and assessed its purity using the Limulus amebocyte lysate assay and by measuring dendritic cell activation.Purified chitosan-based formulations alone failed to induce production of the proinflammatory cytokines tumour necrosis factor alpha (TNF-α) and interleukin (IL)-6 in bone marrow-derived dendritic cells (BMDCs) generated from C57BL/6 mice, while maintaining its ability to promote IL-1β secretion in combination with the Toll-like receptor (TLR)-9 agonist, CpG. Moreover, BMDCs from C3H/HeN and TLR4-deficient mice, C3H/HeJ were stimulated with endotoxin-free chitosan-based formulations and no differences were observed in IL-6 and IL-1β secretion, excluding the involvement of TLR-4 in the immunomodulatory effects of chitosan.The developed method provides simple guidelines for the production of endotoxin-free chitosan, ideal for biomedical applications.