Abstract Airborne particles, particularly those smaller than 2.5 µm, can cause adverse health effects. The health impact of exposure to particles can be magnified when the particles are biological in nature. As an example, exposure to Bacillus anthracis (Ba) can result in death, thus, knowledge of Ba transport characteristics and fate in the environment is critical. Bacillus species form highly resistant endospores that remain viable in the environment for many years until contact with favorable environmental conditions, and they can disseminate through aerosolization. Here, we have developed and tested methods to generate and characterize a surrogate for Ba, Bacillus thuringiensis. In our tests, we cultured and purified spores (>90% purity) in a BSL-2 facility, and used nebulization to aerosolize them. In the first step, we optimized the purification process by characterizing the relative distributions of spores and cells in the different steps of the process. We explored the aerosolized particle characteristics under different spore suspension concentrations. Under low concentrations of the suspension, the mean aerodynamic diameter of single Bacillus thuringiensis spores was 0.72 µm. At higher concentrations of the suspensions, spore clusters with aerodynamic diameters as large as 2 µm were observed. Spore and spore cluster sizes were confirmed with Six-stage Viable Andersen cascade impactor-based measurements. Spore viability during nebulization and deposition was investigated using fluorescent stains acridine orange (all cells) and propidium iodide (permeabilized cells). The validated spore generation process is being used in experiments to deposit single spores and clusters of different sizes on substrates. The objective is to determine forces required to remove these biological particles from substrates of different compositions.