Abstract: There are provided systems and methods for characterization of an assay from a plurality of regions of interest (ROI). The method including: receiving image data of the assay from the plurality of ROI, the image data including at least two color channels for each ROI; determining a ratio of signal change across the color channels for each ROI; converting the ratio of signal change for each well to a concentration measurement of the assay using a calibration curve, the calibration curve determined from image data of a calibration assay with known concentrations; and outputting the concentration measurement for each ROI.