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With international concern growing over the potential for chemical and biological terrorism, there is an urgent need for a sensor that can quickly and accurately detect chemical and biological agents. Such a sensor needs to be portable, robust, and sensitive, with fast sample analysis time. We will demonstrate the use of a micromachined differential mobility spectrometer (DMS) with these characteristics that can detect multiple agents simultaneously on a time scale of seconds. In this study, we have demonstrated the ability of the DMS to detect Bacillus subtilis spores, a surrogate for Bacillus anthracis spores, the causative agent of anthrax. Pyrolysis was used as the sample introduction method to volatilize the spores before introducing material into the DMS. Additionally, we examined the effect of pyrolysis on B. subtilis spores suspended in sterile water using SDS-PAGE. These experiments showed that the spores must be heated at 650°C or greater for 5 s or at 550°C for at least 10 s to be fragmented into particles considerably smaller than 10 kDa, which the DMS can detect. Several major biomarkers can be easily distinguished above the background of the sterile water in which the spores are suspended, and we hypothesize that additional biomarkers could be liberated by further optimizing conditions. The DMS also has shown promise as a detector for chemical weapon agents, and we have demonstrated the ability of the DMS to detect nerve and blister agent simulants at clinically relevant levels.