Methods are provided for the automated detection of assay-positive assay areas in a microfluidic device. When assays are performed in a microfluidic device, the configuration of the microfluidic circuit and its constituent circuit elements can determine where the reagents/analytes used in the assay can be located within the microfluidic circuit. Methods are provided for automatic identification of the size and shape of the assay areas based on a number of parameters which may include type of assay involved, shape and dimensions of microfluidic circuit elements, velocity and physical characteristics of the fluidic medium within the microfluidic circuit, physical/chemical properties of the analytes/reagents, and/or the number of cells being assayed.