Kiton red 620 (KR620) doped polystyrene latex microspheres (PSLs) were synthesized via soap-free emulsion polymerization to be utilized as a relatively nontoxic, fluorescent seed material for airflow characterization experiments. Poly(styrene-co-styrenesulfonate) was used as the PSL matrix to promote KR620 incorporation. Additionally, a bicarbonate buffer and poly(diallyldimethylammonium chloride), polyD, cationic polymer were added to the reaction solution to stabilize the pH and potentially influence the electrostatic interactions between the PSLs and dye molecules. A design of experiments (DOE) approach was used to efficiently investigate the variation of these materials. Using a 4-factor, 2-level response surface design with a center point, a series of experiments were performed to determine the dependence of these factors on particle diameter, diameter size distribution, fluorescent emission intensity, and KR620 retention. Using statistical analysis, the factors and factor interactions that most significantly affect the outputs were identified. These particles enabled velocity measurements to be made much closer to walls and surfaces than previously. Based on these results, KR620-doped PSLs may be utilized to simultaneously measure the velocity and mixing concentration, among other airflow parameters, in complex flows.