Aims: To apply specific collection techniques and spectroscopy to differentiate between live and dead Escherichia coli O157:H7 cells, as well as cells subjected to various inactivation treatments, including heat, salt, UV, antibiotics and alcohol. Methods and Results: Fourier transform-infrared (FT-IR) spectroscopy was used to analyse E. coli O157: H7 cells, after filtration or immunomagnetic collection. Partial least squares analysis of the spectra quantified live E. coli O157: H7 in the presence of dead cells with an R-2 > 0 996. Canonical variate analysis (CVA) not only differentiated between spectra of 100% dead and 100% live cells but also between 1% live : 99% dead and 100% dead. CVA using principal components also differentiated between the spectra of the differentially treated cells at a 95% confidence level, and Cooman plots showed clear separation between clusters of spectra of bacteria exposed to the different inactivation treatments. Mahalanobis distances (MD) corroborated the results of CVA. Conclusions: These results demonstrated the effectiveness of rapid cell collection and FT-IR spectroscopy techniques to differentiate between live and dead E. coli O157: H7 cells. Significance and Impact of the Study: This technique has potential applications for use with foods subjected to various inactivation treatments.