Shewanella oneidensis is able to conserve energy for growth by reducing a wide variety of terminal electron acceptors during anaerobic respiration, including several environmentally hazardous pollutants. This bacterium employs various electron transfer mechanisms for anaerobic respiration, including cell-bound reductases and secreted redox mediators. The aim of this study was to develop rapid tools for profiling the key metabolic changes associated with these different growth regimes and physiological responses. Initial experiments focused on comparing cells grown under aerobic and anaerobic conditions. Fourier transform infrared (FT-IR) spectroscopy with cluster analysis showed that there were significant changes in the metabolic fingerprints of the cells grown under these two culture conditions. FT-IR spectroscopy clearly differentiated cells of S. oneidensis MR-1 cultured at various growth points and cells grown using different electron acceptors, resulting in different phenotypic trajectories in the cluster analysis. This growth-related trajectory analysis is applied successfully for the first time, here with FT-IR spectroscopy, to investigate the phenotypic changes in contrasting S. oneidensis cells. High-performance liquid chromatography (HPLC) was also used to quantify the concentrations of flavin compounds, which have been identified recently as extracellular redox mediators released by a range of Shewanella species. The partial least-squares regression (PLSR) multivariate statistical technique was combined with FT-IR spectroscopy to predict the concentrations of the flavins secreted by cells of S. oneidensis MR-1, suggesting that this combination could be used as a rapid alternative to conventional chromatographic methods for analysis of flavins in cell cultures. Furthermore, coupling of the FT-IR spectroscopy and HPLC techniques appears to offer a potentially useful tool for rapid characterization of the Shewanella cell metabolome in various process environments.