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One concern with current techniques of UV reactor validation is that they provide only a measure of the mean UV fluence. In this research, the actual fluence distribution of a UV reactor is measured through the use of photochemically active fluorescent microspheres. Experimental tests were performed in a pilot-scale monochromatic UV 254 nm reactor operated at two flow rates. Analysis of the fluorescence intensity decay was performed using collimated beam experiments for determination of decay rate kinetics. A stochastic hierarchal process involving Bayesian statistics, and the Markov chain Monte Carlo integration technique was used to correlate the microsphere fluorescence intensity distribution to the UV fluence distribution. The experimental UV fluence distribution was compared with the fluence distribution predicted using a computational fluid dynamics model. The results showed that the fluorescent microspheres measured a wider distribution of UV fluences with a flow rate of 3 gpm than with 7.5 gpm. The principal differences between the modeled and the measured distribution were in the low UV fluences where the microspheres predicted lower fluence levels than the model. The use of microspheres is demonstrated as a novel technique for measurement of the fluence distribution in UV reactors. This technique has both fundamental and practical implications for reactor evaluation and testing and could improve confidence in the future use of mathematical models for UV reactor characterization. It also serves as a complement to biodosimetry testing by providing greater insights regarding reactor behavior and validation.