Raman micro-spectroscopy can be used to investigate biological single cells exposed to different chemicals. Since chronic exposure at low doses of pesticides can promote several diseases, the investigation of cellular changes induced by exposure to non-cytotoxic doses of pesticides is of increasing interest. The efficiency of Raman micro-spectroscopy to detect chemical modification in normal human keratinocytes induced by exposure to non-cytotoxic doses of chlorpyriphos, an organophosphate pesticide present in many plant-protection products, was investigated. Such modification affects mainly proteineous components (both single amino acids and amide linkages between amino acids) of the nucleus, cellular membranes and cytoplasm as well as the nucleic acid component of the nucleus. Chemical modifications are already detectable after 24 h exposure of keratinocytes at a chlorpyriphos concentration of 10−6M, which is three orders of magnitude lower than the cytotoxic concentration (10−3M). Heavy damage to the lipid component occurs after exposure to the nearly cytotoxic concentration (10−4M). Atomic force microscopy images of keratinocyte cells exposed for 24 h to various chlorpyriphos concentrations show a progressive deterioration of the morphology of cellular membrane as the chlorpyriphos concentration increases. The results of this work may have wide applications in the monitoring of molecular changes in single human cells exposed to toxic agents. Copyright © 2010 John Wiley & Sons, Ltd.