The aim of this study was to evaluate the impact of the absence of water during the synthesis of thiolated poly(acrylic acid) (PAA) on its mucoadhesive properties. Previously, thiolation of hydrophilic polymers was performed in aqueous media, where (poly)acrylates undergo a conformational transition to form an entangled network of polymer chains stabilized by neutralization. The impact of chain entanglement, porosity and degree of functionalization of anhydrous precipitated polymers on their mucoadhesive characteristics were therefore evaluated in comparison to the equivalent products synthesized in water and dried by lyophilization.Anhydrous synthesis and purification procedures were designed for the thiolation and entire preactivation of PAA by means of immobilization of l-cysteine (PAA-Cys) and 2-mercaptonicotinic acid (PAA-Cys-MNA), respectively. The obtained conjugates were characterized chemically and for their particle size. Differences in mucoadhesive properties were determined by means of the residence time as well as tensile strength of polymer test disks on porcine small intestinal mucosa.Anhydrous products revealed a 4.2-fold higher amount of immobilized free thiol moieties for PAA-Cys and a 3.1-fold higher quantity of 2-mercaptonicotinic acid for PAA-Cys-MNA. The impact of particle size of anhydrous products on their mucoadhesive properties was negligible. In comparison to the corresponding aqueous products a 2.5-fold increase in residence time for PAA-Cys and 21.2-fold increase for PAA-Cys-MNA was achieved and in agreement with these results, the improvement in the total work of adhesion was 2.9-fold for PAA-Cys and 5.1-fold for PAA-Cys-MNA.According to these findings, anhydrous synthesis conditions seem to be a promising tool for improved modification rates and moreover enhanced mucoadhesive properties of hydrophilic polymers.