A novel methodology has been developed for the determination (i.e., identification and quantification) of bacterial spores that may be useful in many applications; most notably, development of detection schemes toward potentially harmful biological agents such as Bacillus anthracis. In addition, this method would be useful as an environmental warning system where sterility is of importance (i.e., food preparation areas as well as invasive and minimally-invasive medical applications). This method is based on the infrared (1500 to 4000-nm) absorption of fatty acids and peptides extracted from the spore. The absorption spectra of several bacterial spore extracts in carbon disulfide solution have been measured. Further, the groups of absorption bands in this region are unique for each spore, which implies it may be possible to use this technique for their determination. The Bacillus spores studied were chosen because they are taxonomically close to each other as well as to Bacillus anthracis. Expectedly, the measured absorption bands are heavily overlapped since the extracted analytes are similar in structure for each Bacillus spore. Additionally, this makes it impossible to use a single wavelength for the determination of any bacterial spore species. However, it may be possible to use the infrared absorption technique in conjunction with the Partial Least Squares (PLS) regression method to develop statistical models for the determination of bacterial spores. Results will be presented concerning sampling, data treatment, and development of PLS models as well as application of these models in the determination of unknown Bacillus bacterial spores.