Pressure-assisted thermal processing (PATP) is being widely investigated for processing low acid foods. However, its microbial safety has not been well established and the mechanism of inactivation of pathogens and spores is not well understood. Fourier transform infrared (FT-IR) spectroscopy was used to study some of the biochemical changes in bacterial spores occurring during PATP and thermal processing (TP). Spore suspensions (∼109 CFU/mL of water) of Clostridium tyrobutyricum, Bacillus sphaericus, and three strains of Bacillus amyloliquefaciens were treated by PATP (121°C and 700 MPa) for 0, 10, 20, and 30 s and TP (121°C) for 0, 10, 20, and 30 s. Treated and untreated spore suspensions were analyzed using FT-IR in the mid-infrared region (4000-800 cm-1). Multivariate classification models based on soft independent modeling of class analogy (SIMCA) were developed using second derivative-transformed spectra. The spores could be differentiated up to the strain level due to differences in their biochemical composition, especially dipicolinic acid (DPA) and secondary structure of proteins. During PATP changes in α-helix and β-sheets of secondary protein were evident in the spectral regions 1655 and 1626 cm-1, respectively. Infrared absorption bands from DPA (1281, 1378, 1440, and 1568 cm-1) decreased significantly during the initial stages of PATP, indicating release of DPA. During TP changes were evident in the bands associated with secondary proteins. DPA bands showed little or no change during TP. A correlation was found between the spore's Ca-DPA content and its resistance to PATP. FT-IR spectroscopy could classify different strains of bacterial spores and determine some of the changes occurring during spore inactivation by PATP and TP. Furthermore, this technique shows great promise for rapid screening PATP-resistant bacterial spores. © 2007 American Chemical Society.