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Accurate and rapid analytical methods are essential for the detection and identification of biological warfare agents (BWA). Although various studies have investigated the use of MALDI-TOF MS for bacterial classification, only a few studies have examined the applicability of method for the identification of BWAs. This study aimed to generate, collect and analyse Bacillus spore aerosol particles of 2–10 μm, the optimal size of a BWA. Bacillus spore aerosol particles of 2–10 μm were rapidly analysed using direct in situ MALDI-TOF MS without pretreatment processes. A mass spectrum of Bacillus spore aerosol particles was successfully detected. For real-time detection and identification, a mass spectral database of Bacillus spores was constructed, and an algorithm was developed and applied. Bacillus spore particles were rapidly detected and identified by MS, which can be used for the detection and inspection of BWAs.Significance and Impact of the Study: Methods that permit accurate and fast detection and identification are essential in the case of pathogens such as Bacillus anthracis, which can be used as biological warfare agents or bioterrorism materials. Here, direct in situ MALDI-TOF MS allowed the high-throughput detection and identification of aerosolized Bacillus spore particles, and this method could be effectively applied for the detection and inspection of BWAs. In this context, we believe that this method may contribute to the realization of real-time and on-site identification of BWAs.