Oxidative stress is encountered in many biological systems; the resultant oxidative injury plays a significant role in the pathogenesis of diverse diseases. Conventional measurements on oxidative injury are employed almost exclusively on a large population of cells either by counting the fraction of cell death or by observing the fluorometric change resulting from exogenous reagents, thereby lacking in molecular detail and temporal specificity. In this work we combine laser tweezers and Raman spectroscopy to observe the response of single cells to oxidative stress. By measuring the temporal changes of vibrational spectra of single optically trapped cells, we demonstrate a molecular-level assessment of cellular oxidative injury in real time, both qualitatively and quantitatively, without the introduction of exogenous reagents. The main experimental findings are supported by the observation of Raman spectra of intermediates and downstream products. The abrogation of the above changes by ascorbic acid further illustrates the therapeutic effect of antioxidants against cellular oxidative injury. This approach is extensible to studies exploring the biochemical transformation of single cells or intracellular organelles in response to various chemical or physical stimuli. With the aid of ‘molecular fingerprints’, single-cell Raman spectroscopy exhibits a great potential for accessing the chemical aspects of cellular bioactivity, yielding insight into pathophysiological processes and assisting the development of novel therapeutic interventions against diseases. Copyright © 2009 John Wiley & Sons, Ltd.