A denatured avidin-bonded column was suitable for use in the reversed-phase HPLC and complete recovery of serum protein over a wide range of pH. Retention property of the denatured avidin-bonded column was very nearly to a non-denatured avidin-bonded column already reported and, however, showed very high stability to organic solvent. A performance of the denatured avidin-bonded column was maintained even if 500 continuous injections of human serum (total 10 ml).