The polysaccharide intercellular adhesin ( PIA) confers major functional effects in biofilm formation, which bears an important role in the pathogenicity of Staphylococcus epidermidis. Following the identification of biofilm-forming strains by biochemical and molecular methods, isogenic strain was prepared and in vitro biofilm formation assay was performed consequently. By parallel analysis of both the PIA-positive and PIA-negative strains using size exclusion chromatography by Fast protein liquid chromatography ( FPLC) method, the respective PIA was purified. Recovered PIA was examined using colorimetric and hemagglutination assays. Finally, the recovered PIA was analyzed using Fourier-transform infrared spectroscopy and proton nuclear magnetic resonance spectroscopy methods. By the parallel purification process and comparison of the obtained graphs from the FPLC detector, fractions near the void volume were determined as PIA. The colorimetric and hemagglutination assays were applied and the content of carbohydrates ( hexose = 620 mu g/ml, hexosamine = 5700 mu g/ml and ketoses = 170 mu g/ml) and hemagglutination titer ( 1: 128) in recovered polysaccharide were determined. This study shows that PIA has a significant role in the biofilm formation in S. epidermidis strains. The recovered polysaccharide and its molecular weight were analyzed within the near void volume of the utilized column.