The SC-41 fluorescence Plate Reader enables high-throughput measurements on the FS5 spectrofluorometer via automated measurements of microwell plates. Standard and custom microwell plates can be measured containing up to 384 wells. Both spectral and lifetime measurements can be acquired, a unique feature of the SC-41. This technical note demonstrates the use of the SC-41 Plate Reader for ratio analysis. The emission spectra can be measured from each well where samples contain varying ratios of two fluorophores. The Trend Analysis feature in the Fluoracle software can then be used to plot a calibration curve which allows the determination of the ratio of each fluorophore in unknown samples.