The majority of Escherichia coli O157:H7 outbreaks are associated with ground beef. To detect this pathogen, separation techniques were tested with E. coli O157:H7 in ground beef followed by FT-IR analyses. Ground beef samples were inoculated with various levels of live and heat treated E. coli O157:H7 cells and the bacteria were extracted by filtration or immunomagnetic separation (IMS). Spectra were collected and detection limits were established by discriminant analysis of the 1800-800 cm-1 region and comparison to standard plate counts. The detection limit for the Filtration-FT-IR and IMS-FT-IR assays was 105 CFU/g. Partial least squares model established significant linear relationships between plate counts and spectra [R ≥ 0.99]. Discriminant analysis and canonical variate analysis of the spectra differentiated live and heat treated cells of E. coli O157:H7. Validation experiments using ground beef inoculated with fewer cells (101- 102 CFU/g) reached the detection limit within a six hour incubation. A portable IR sensor was also used to detect E. coli O157:H7 in ground beef, and the detection limit was 107 CFU/g. The total time to detection for Filtration-FT-IR and IMS-FT-IR were one hour and 3.75 h, respectively which is faster than conventional plate count methods (48h) and conventional IMS methods (48h). The FT-IR methods developed are potentially rapid and simple protocols that could be further developed for the detection of different species of pathogenic bacteria in complex food systems.