An evaluation of Fluorescent YG-microspheres (Polysciences Inc.) was performed to simulate Cryptosporidium parvum (C. parvum) oocysts inactivation in treatment systems that utilize multiple disinfectants. Experiments were conducted in batch reactors that included an ozone primary stage and a secondary free chlorine treatment stage. A flow cytometer was used to track changes in the fluorescence intensity distribution due to exposure to the chemical disinfectant. Microsphere 'survival ratios' (N/No) were calibrated by selecting an appropriate fluorescence intensity threshold to replicate the inactivation of different C. parvum oocysts strains. Results showed that fluorescent microspheres displayed synergistic effects in the presence of two sequential disinfectants. In addition, microsphere structural tests showed that the polystyrene surface was damaged due to exposure to ozone. This polystyrene damage enhanced the diffusion of the secondary disinfectant into the microsphere, where dye was degraded in the opened polymer layer. As a result, YG-fluorescent microspheres is a promising non-biological technique that is capable of producing similar synergistic behavior as with C. parvum oocysts exposed to ozone followed by chlorine.