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The work in this dissertation presents a method for multiplexing fluorescence measurements in both polymerase chain reaction (PCR) and anisotropy. For PCR, two dual color infrared mediation assays were demonstrated on a microfluidic device. Infrared mediated PCR on a microchip allowed a 40 cycle assay, which would require 1 hour on a traditional instrument to be reduced to 35 min due to the decrease volume and increased heating and cooling efficiency. A plasmid, PU19, was amplified in the presence of a DNA intercalating dye, EvaGreen™, and a passive reference dye, ROX, with an efficiency of 96%. The ROX signal was used to correct for inter-run and inter-chip variations in excitation volume. A melt curve was taken simultaneously with amplification and showed a single peak at 82 °C corresponding to the known melt temperature of the plasmid. Frequency modulation was used to isolate the fluorescence signals for the EvaGreen™ and the ROX from the large background present due to the tungsten lamp providing infrared light for the heating of the microfluidic device as well as demonstrate the first multi-color infrared quantitative PCR on a microfluidic device. Frequency modulation was used to demonstrate a multi-analyte anisotropy method inflow on a microfluidic device. An anisotropy immunoassay was developed for the simultaneous detection of insulin and glucagon at physiologically relevant levels. Frequency modulation was used to reduce the number of optics required for multi-fluorophore anisotropy measurements as well as increase the signal to noise in the measurement by 20 fold. The increased signal to noise ratio resulting in an improvement in the limit of detection from 10 nM to 50 pM. The anisotropy immunoassay assay was expanded to an online format used to measure the secretion of insulin by islets of Langerhans which were housed on the microfluidic device. The microfluidic system was able to stimulate islet with 3mM and 20 mM glucose and measure the resulting secretions. The temporal resolution of the system was less than 5 minutes and the highly automated fashion in which the online assay function should make it amenable to further islet studies as well as other biological systems.