Several immobilization methods were explored for the preparation of high-performance affinity monolithic columns containing human serum albumin (HSA). These monoliths were based on a copolymer of glycidyl methacrylate and ethylene dimethacrylate. In one method, the epoxy groups of this copolymer were used directly for the immobilization of HSA through its amine residues (i.e., the epoxy method); in other approaches, these epoxy groups were converted to diols for later use in the carbonyldiimidazole, disuccinimidyl carbonate, and Schiff base methods. Each HSA monolith was evaluated in terms of its total protein content and its retention of several model compounds, including (R/S)-warfarin and D/L-tryptophan. The greatest amount of immobilized HSA was obtained by the Schiff base method, whereas the epoxy method gave the lowest protein content. The Schiff base method also gave the best resolution in chiral separations of (R/S)-warfarin and D/L-tryptophan. All of the immobilization methods gave similar relative activities for HSA in its binding to (R)- and (S)-warfarin, but some differences were noted in the activity of the immobilized HSA for D- and L-tryptophan. The efficiency of these monoliths was found to be greater than that of silica-based HSA columns for (R/S)-warfarin (i.e., analytes with high retention), but little or no difference was seen for D- and L-tryptophan (analytes with weak retention).