This invention relates to an assay device and a method for pretreating a specimen containing a biologically-relevant substance and then assaying the biologically-relevant substance. Biologically-relevant substances used in the assay method include microorganisms, cells, viruses, nucleic acids, polysaccharides, proteins (including antigens, antibodies, chromoproteins, and enzymes), peptides, nucleic acids, and small molecules. This pretreatment method removes contaminants from the biologically-relevant substances using supports such as magnetic particles, gels, resins, membranes, and solid-phased reagents. Therefore, in the assay method the following steps are generally carried out (i) a pretreatment step of treating a specimen using one or more first supports and then one or more second supports, and (ii) the assay step carried out after the pretreatment step. In particular, the pretreatment method reduces false positives and false negatives in the assay.