Rheological analyses provided a valuable tool for monitoring the auto-crosslinking reaction of HA TBA salt in organic solvents. ACP intermediates, powders and hydrogels were characterized, elucidating important information on the crosslinking process. A twostep mechanism was proposed to explain the trends in viscosity and particle size. Reaction parameters were varied to investigate the influence of solvent and temperature, and it was found that varying these parameters did not modify the crosslinking degree appreciably but did greatly influence the intra-to intermolecular crosslinking ratio and, subsequently, the viscoelastic properties. ESEM analysis revealed a compact and sheet-like matrix structure of the ACP powders obtained at lower temperature and in the presence of NMP and DMF. The same ACP powders formulated as hydrogels exhibited stronger viscoelastic properties. The hyaluronidase assay afforded an unexpected result: the enzymatic cleavage rate constant of ACP, at low elastic modulus, was lower than that of other, chemically more resistant, crosslinked HAs (i.e., HDC and HBC). Further in vivo studies may be performed, considering that the presence of other enzymes (e.g., esterases) could affect this outcome.