A method of compensation for spectral crosstalk in multiplex nucleic acid amplification, comprising: a calibration process comprising performing at least a series of single-plex nucleic acid amplification of a nucleic acid sample using a detection probe, wherein the nucleic acid amplification occurs over a plurality of interrogation periods; from the nucleic acid amplification, acquiring amplification data that indicates an amount of nucleic acid present for each of the plurality of interrogation periods; and based on the amplification data, determining a crosstalk correction value associated with a near spectral neighbor to the detection probe to reduce spectral crosstalk from the near spectral neighbor; the method further comprising nucleic acid amplification analysis including applying the crosstalk correction value obtained from the calibration process to amplification data collected from multiplex nucleic acid amplification of nucleic acid samples, wherein the determined crosstalk correction value is the ratio of a signal detected for the near spectral neighbor to the signal detected for the amplification data of the detection probe, wherein the near spectral neighbor is determined based on a distance from a fluorescence peak of the target dye of the detection probe to the fluorescence peak of a neighboring target dye, wherein the fluorescence peak of the target dye of the detection probe and the fluorescence peak of the target dye of the near spectral neighbor are separated by a distance less than a threshold distance, and wherein applying the crosstalk correction value to the amplification data comprises: subtracting a background signal from the signal detected for the amplification data to obtain a background-corrected signal; and subtracting, from the background-corrected signal, a product of the crosstalk correction value and the signal detected by the near spectral neighbor. a calibration process comprising performing at least a series of single-plex nucleic acid amplification of a nucleic acid sample using a detection probe, wherein the nucleic acid amplification occurs over a plurality of interrogation periods; from the nucleic acid amplification, acquiring amplification data that indicates an amount of nucleic acid present for each of the plurality of interrogation periods; and based on the amplification data, determining a crosstalk correction value associated with a near spectral neighbor to the detection probe to reduce spectral crosstalk from the near spectral neighbor; the method further comprising nucleic acid amplification analysis including applying the crosstalk correction value obtained from the calibration process to amplification data collected from multiplex nucleic acid amplification of nucleic acid samples, wherein the determined crosstalk correction value is the ratio of a signal detected for the near spectral neighbor to the signal detected for the amplification data of the detection probe, wherein the near spectral neighbor is determined based on a distance from a fluorescence peak of the target dye of the detection probe to the fluorescence peak of a neighboring target dye, wherein the fluorescence peak of the target dye of the detection probe and the fluorescence peak of the target dye of the near spectral neighbor are separated by a distance less than a threshold distance, and wherein applying the crosstalk correction value to the amplification data comprises: subtracting a background signal from the signal detected for the amplification data to obtain a background-corrected signal; and subtracting, from the background-corrected signal, a product of the crosstalk correction value and the signal detected by the near spectral neighbor. <EOS>