Haptoglobin is known to bind to hemoglobin during intravascular hemolysis. Membrane-immobilized anti-haptoglobin antibody, which was produced after antibody was isolated by non-denaturing two-dimensional electrophoresis, was transferred to a polyvinylidene difluoride membrane and was stained using Ponceau S. The proteins bound to the membrane-immobilized anti-haptoglobin antibody were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. Hemoglobin was specifically obtained when the membrane-immobilized anti-haptoglobin antibody was incubated with human serum obtained from hemolysis blood. Furthermore, hemoglobin in the flowing fluid was captured by the membrane-immobilized anti-haptoglobin antibody and analyzed directly. The results indicate that hemolysis can be examined by direct trapping and analysis of hemoglobin under physiological conditions.