A subtractive immunoassay for detecting the presence or absence of first and second analytes in a sample includes in the following order the steps of: a) conjugating a first detector antibody to the first analyte to tag the first analyte with a first detector tag; b) conjugating a first capture antibody to the first analyte to capture the first analyte to completely deplete the first analyte from the sample; c) conjugating a second detector antibody to the second analyte in the depleted sample to tag the second analyte with a second detector tag; d) conjugating a second capture antibody to the second analyte to capture the second analyte from the sample; e) detecting presence or absence of the first and second detector tags at the site of the first and second capture antibody, respectively, and thereby detecting the presence or absence of the first and second analytes in the sample.