The study was aimed to synthesize preactivated polysulfonate thiomers using poly(4-styrenesulfonic acid-co-maleic acid) (PSSA-MA) and to evaluate their permeation enhancing properties. PSSA-MA-cysteamine (PC) conjugates with 2-mercaptonicotinic acid (2MNA) having different degree of preactivation (PC1608-2MNA, PC2300-2MNA, PC3100-2MNA) were synthesized from the subsequent PSSA-MA-cysteamine thiomers (PC1608, PC2300 and PC3100). The permeation-enhancing features were evaluated by in-vitro models using low-molecular size marker sodium fluorescein (Na-Flu) and with high-molecular size marker fluorescein isothiocyanate-dextran (FD4). Associating the influence of degree of preactivation on permeation enhancement, following rank order PC3100-2MNA > PC3100 > PC2300-2MNA > PC2300 > PC1608-2MNA > PC1608 > PSSA-MA > control was observed on Caco-2 cell monolayers and with little change in sequence on freshly excised rat intestine. The apparent permeability (Papp) was improved 3.16-fold for Na-Flu and 3.51-fold for FD4 on Caco-2 cell monolayers. Similarly, 4.17- and 3.60-fold improved Papp values were observed on freshly excised rat intestine for Na-Flu and FD4, respectively. More pronounced permeation effects on rat intestine compared to Caco-2 cell monolayer by the thiomer/preactivated conjugates indicated their mucus-interpenetration capability in addition to mucoadhesion. Thus relatively low molecular weight (LMW) preactivated polysulfonate thiomers could to a higher extent enhance permeation on membranes covered by mucus layer compared to high molecular weight thiomers/preactivated conjugates that usually exhibit higher permeation enhancing effects on Caco-2 cell monolayer.