Abstract: The present disclosure relates methods of chromogen layering, wherein a first chromogen and a first stain (color) are produced on a sample, specific for a first analyte, followed by a second chromogen and a second stain (color) being produced on the same sample, specific for a second analyte. In addition, if desired, by overlaying the second stain on top of the first stain, a unique third color is produced that is specific for a third analyte. Therefore, the distribution of different colors throughout the sample could be used to identify at least two or more analytes simultaneously within a single sample.