Calibrating and verifying 2-D and 3-D vadose zone flow and transport models requires detailed information on water and solute redistribution. Among the different water flow and mass transfer determination methods, staining tracers have the best spatial resolution allowing visualization and quantification of fluid flow including preferential flow paths. Staining techniques have been used successfully for several decades; however, the hydrological community is still searching for an “ideal” vadose zone tracer regarding flow path visualization. To date, most research using staining dyes is carried out with Brilliant Blue FCF. Fluorescent dyes such as Uranine, however, have significant advantages over nonfluorescents which makes them a promising alternative. This paper presents the first analysis of key properties any fluorescent substance must possess to qualify as a staining fluorescent tracer in vadose zone hydrological applications. First, we summarize the main physico-chemical properties of Uranine and evaluate its staining tracer potential with conventional suitability indicators and visibility testing in a soil profile. Based on numerical analysis using the theory of fluorescence, we show that a low molar absorption coefficient is a crucial parameter to quantify concentration accurately. In addition, excitation of a tracer on wavelengths different from the maximum excitation wavelength can extend the linear range of the concentration-fluorescence relationship significantly. Finally, we develop criteria for evaluating the suitability of any potential fluorescent soil staining compound for soil hydrological applications: 1) high quantum yield, 2) low molar absorption coefficient, 3) fluorescence independent of temperature, 4) low photodecomposition rates, and 5) fluorescence stable across a wide range of pH values.