In one exemplary embodiment, a method for validating an instrument is provided. The method includes receiving amplification data from a validation plate to generate a plurality of amplification curves. The validation plate includes a sample of a first quantity and a second quantity, and each amplification curve includes an exponential region. The method further includes determining a set of fluorescence thresholds based on the exponential regions of the plurality of amplification curves and determining, for each fluorescence threshold of the set, a first set of cycle threshold (Ct) values of amplification curves generated from the samples of the first quantity and a second set of Ct values of amplification curves generated from the samples of the second quantity. The method includes calculating if the first and second quantities are sufficiently distinguishable based on Ct values at each of the plurality of fluorescence thresholds.