A method was established using hollow fiber-liquid phase microextraction(HF-LPME) followed by high performance liquid chromatography(HPLC) to determine the concentration of the free(unbound) drug in the solution of the drug and protein. Measurements of drug-protein binding ratios and free drug concentrations were then analyzed with the Klotz equation to determine the equilibrium binding constant and number of binding sites for drug-protein interaction. The optimized method allows one to perform the efficient extraction and separation of free drug from protein-bound drug, protein, and other interfering substances. This approach was used to characterize the binding of the anticholinergic drugs atropine sulfate and scopolamine hydrobromide to proteins in human plasma and bovine serum albumin(BSA). The results demonstrate the utility of HF-LPME method for measuring free drug concentrations in protein-drug mixtures and determining the protein binding parameters of a pharmacologically important class of drugs. A method was established using hollow fiber-liquid phase microextraction (HF-LPME) followed by high performance liquid chromatography (HPLC) to determine the concentration of the free (unbound) drug in the solution of the drug and protein. Measurements of drug-protein binding ratios and free drug concentrations were then analyzed with the Klotz equation to determine the equilibrium binding constant and number of binding sites for drug-protein interaction. The optimized method allows one to perform the efficient extraction and separation of free drug from protein-bound drug This approach was used to characterize the binding of the anticholinergic drugs atropine sulfate and scopolamine hydrobromide to proteins in human plasma and bovine serum albumin (BSA). The results demonstrates the utility of HF-LPME method for measuring free drug concentrations in protein-drug mixtures and determining the protein binding parameters of a pharmacologically import ant class of drugs.