Abstract: Determination of exact total protein bonding quantity is often a key step in the preparation of protein-immobilized chiral monolith. In this study, we developed and evaluated a bovine serum albumin(BSA)modified monolith based on glycidyl methacrylate(GMA)and ethylene dimethacrylate(EDMA)for chiral separation. The epoxy groups of the polymer were used directly for the covalent bonding of BSA. A Coomassie brilliant blue(CBB)protein assay(Bradford method)was established to determine the protein bonding quantity, and the influence of some key aspects such as ionic strength, pH value and reaction time were studied. The method was validated with respect to linearity, precision, accuracy and robustness. The maximum amount of immobilized BSA was 11. 90 mg/g, obtained using 65 ∶35 cyclohexanol/dodecanol as the porogen. The monolith was successfully applied in the chiral separation of R/S-warfarin and D/L-tryptophan in only 1-20 min. Furthermore, the chromatographic conditions like pH and organic additive of the mobile phase were optimized. The chiral separation performance of this BSA-immobilized monolith is positively correlated to the protein bonding quantity.