Cystic fibrosis (CF) is a genetic disorder with serious health implications if not diagnosed early in life. The current diagnostic test is the sweat test, using chloride ion concentration to determine the patient’s diagnosis. This research is focused on an alternative diagnostic method using a system of chloride ionophore and polarity-sensitive dye (PSD). Polarity-sensitive dyes, in combination with chloride ionophores and sweat, have a visible color shift; PSDs also fluoresce in specific environmental conditions. Here, we investigate the fluorescence of Nile red, Nile blue, and DiA to examine its effectiveness in detecting small changes in the ion concentration of biological solutions. Both Nile blue and Nile red have shown color change and fluoresce in different chloride ion concentrations, with potential uses in diagnosing CF.