A biochemical approach has been used to analyze the surface chemistry of a hydrated polyester-urea-urethane. Using an enzyme-catalyzed hydrolysis reaction, the surface chemistry of the polyurethane was probed by having the enzyme remove the polymer chain components making up the surface. The surface-derived products were analyzed using high performance liquid chromatography (HPLC), and product identification was carried out by mass spectrometry. The qualitative and quantitative analysis of the removed surface-derived products provided the necessary information required to reconstruct the chemistry of the hydrated polymer surface. The results indicated that methane and urea linkages were present on the hydrated surface. However, their concentrations were lower than in the bulk polymer. In addition, the ratio of urea to methane groups was substantially reduced at the hydrated surface relative to the bulk polymer stoichiometry. The data obtained for the hydrated surface were also compared to the analysis of the dry surface, carried out using X-ray photoelectron spectroscopy (XPS). The hydrated polymers contained higher urethane and ester groups than that shown on the dry surface. While XPS was able to identify the presence of urethane linkages on the dry surface, these could not be accurately quantified. Furthermore, the content of urea linkages could not be specifically determined by XPS because they were masked by the ester groups.