A method of quantitatively analyzing an analyte contained in a whole blood sample, wherein a dry multi-layered analysis element is used. The method provides particular merits when the used multi-layered analysis element has no light-shielding layer which is interposed, in the conventional analysis element, between a coloring reagent layer and a blood cell separating layer, so that red coloring matters of blood cells are detected from the support side during the step of measuring the optical density of the reflected light. After the optical density due to coloring matters of blood cells trapped by the blood cell separating layer has reached a constant level or background density, the changing rate in optical density is measured and then the measured changing rate is converted to the corresponding content or activity of the analyte through a colorimetric operation, or the total change in optical density is measured and then the substantially constant background density is subtracted therefrom to know the change in optical density caused by a coloring dye or like material formed in the coloring reagent layer in the presence of the analyte, followed by a similar conversion operation performed on the basis of the principle of colorimetry, to determine the content or activity of the analyte.