The ultraviolet (UV) Fluorescent Aerodynamic Particle Sizer (FLAPS), a flow cytometer-like apparatus was developed by the Canadian Department of National Defence for real-time detection of autofluorescence of biological aerosol particles such as bacterial spores. The direct relation between autofluorescence intensity and viability has recently been reported and viable spore are more autofluorescent in UV (Laflamme, Frontiers in Bioscience). The goal of this manuscript is to describe a flow cytometry sorting protocol based on UV-induced autofluorescence. An EPICS ELITE ESP flow cytometer equipped with a UV laser and cell sorter was used to mimic the optical properties of FLAPS and to study the two extremes of a spore population according to its autofluorescence (lower level of autofluorescence (LLA) and higher level of autofluorescence (HLA) spores). Bacillus subtilis var niger was used as a surrogate for Bacillus anthracis spores and sorted using autofluorescence intensity as the main criterion. The protocol developed in our laboratory to sort Bacillus spores according to their autofluorescence properties is described. Purity of each sorted population was greater than 95%. Using autofluorescence as the main criterion, we demonstrate that it is possible to separate two distinct spore populations.