At Lawrence Livermore National Laboratory, we have developed a single-particle aerosol mass spectrometry (SPAMS) system that can rapidly analyze individual micrometer-sized biological aerosol particles or cells that are sampled directly from air or a lab-generated aerosol into a mass spectrometer. As particles enter the SPAMS system, their aerodynamic size and fluorescence properties are measured before mass spectra from both positive and negative ions created by matrix-free laser desorption and ionization are recorded. All the correlated data obtained from a particle can be analyzed and classified in real-time. The SPAMS system is capable of discriminating, particle by particle, between bacterial spores, vegetative cells and other biological and non-biological background materials using the mass fingerprints obtained from those particles. In addition, selected species of bacteria can be discriminated from each other with this method. Here we describe the overall architecture of the SPAMS system and the related algorithms. We present selected results from applying the SPAMS technique to the analysis of biological agent simulants and single cells. We also describe results from first proof-of-concept experiments using SPAMS for the rapid screening of human effluents for tuberculosis. Lastly, we present results from a field study in a large airport using SPAMS to assess biological content in ambient aerosol.