Investigating the relative efficiencies of molecular chaperones is important for understanding protein biosynthesis inside a cell. We developed an analytical method for estimating relative chaperone activity under physiological, multi-chaperone conditions using a protein-conjugated column. A chaperone mixture was subjected to chromatography on a column conjugated with denatured ovalbumin, and the elution positions of target chaperones were compared using western blotting to determine the relative affinity of each chaperone for the denatured protein. Because molecular chaperones should be eluted according to their strength of association with the denatured ovalbumin in the column, the elution position must accord with the chaperone activity and can be used as an indicator of relative chaperone activity. We found that the column procedure was effective in an assay of a mixture of calreticulin and BiP, the molecular chaperones in the endoplasmic reticulum; the assay showed that calreticulin associated with denatured ovalbumin more strongly than BiP.