Confocal Raman micro-spectroscopy (CRMS) was used to measure time-course spectral images of live cells undergoing apoptosis without using molecular labels or other invasive procedures. Human breast cancer cells (MDA-MB-231) were exposed to 300 µM etoposide to induce apoptosis, and Raman spectral images were acquired from the same cells at 2-h intervals over a period of 6 h. The purpose-built inverted confocal Raman micro-spectrometer integrated an environmental enclosure and wide-field fluorescence imaging. These key instrumental elements allowed the cells to be maintained under sterile physiological conditions (37 °C, 5% CO2) and enabled viability and apoptosis assays to be carried out on the cells at the end of CRMS measurements. The time-course spectral images corresponding to DNA Raman bands indicated an increase in signal intensity in apoptotic cells, which was attributed to DNA condensation. The Raman spectral images of lipids indicated a high accumulation of membrane phospholipids and highly unsaturated non-membrane lipids in apoptotic cells. This study demonstrates the potential of CRMS for label-free time-course imaging of individual live cells. This technique may become a useful tool for in vitro toxicological studies and testing of new pharmaceuticals, as well as other time-dependent cellular processes, such as cell differentiation, cell cycle and cell–cell interactions. Copyright © 2010 John Wiley & Sons, Ltd.