There is a need for time efficient evaluation methods to discriminate between viable and dead bacterial spores. In this work, the potential to use the autofluorescence from spore suspensions for evaluation of spore deactivation processes is investigated. Bacillus thuringiensis and Bacillus anthracis ATCC 4229 spores were exposed to UV-radiation for deactivation and the fluorescence response was monitored at different radiation doses and the deactivation was evaluated via traditional bacterial incubation on agar culture plates. For excitation wavelengths of, e.g., 280 m and 330 nm, differences in the fluorescence response could be observed for different live:dead ratios.