This article described the development of a simple method for quantifying the flow of fluids in paper microfluidic devices with varying fluidic channel thicknesses. Microfluidic channels in paper were formed when the hydrophilic paper was patterned with a photosensitive polymer or wax materials that created hydrophobic walls through the entire thickness of the paper. Fluid distribution rates through the paper were increased with the decreasing thickness of the hydrophilic channels. The developed paper microfluidic device exhibited a consistent linear relationship between the wicked distance in the paper and the volume of fluids. The sensitivity of the device was as low as 0.2 μL of the testing fluid volume. Based on the consistent performance of quantifying fluid volume, the paper microfluidic device would enable more accurate and sensitive diagnosis than current dry eye tests that use capillary force for liquid imbibitions.