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Microfluidic fluorescence assay devices show great promise as preclinical and clinical diagnostic instruments. Normally, fluorescence signals from microfluidic chips are quantified by analysis of images obtained with a commercial fluorescence microscope. This method is unnecessarily expensive, time consuming, and requires significant operator training, particularly when considering future clinical translation of the technology. In this work, we developed a dedicated low cost fluorescence microfluidic device reader (FMDR) to read sandwich immunofluorescence assay (sIFA) devices configured to detect vascular endothelial growth factor ligand concentrations in ocular fluid samples. Using a series of sIFA calibration standards and a limited set of human ocular fluid samples, we demonstrated that our FMDR reader has similar sensitivity and accuracy to a fluorescence microscope for this task, with significantly lower total cost and reduced reading time. We anticipate that the reader could be used with minor modifications for virtually any fluorescence microfluidic device.