Surface-enhanced Raman spectroscopy (SERS) has found increasing acceptance in art conservation and forensic science for its great potential in detecting trace amounts of material. However, SERS is not a separation technique, and, therefore, it is not always suitable for distinguishing different components in a mixture. Coupling of thin layer chromatography (TLC) and SERS has been investigated in this article as a tool for the separation and identification of four alkaloids, namely harmalol, harmaline, harmane and harmine, from the seed extract of Syrian rue (Peganum harmala). The alkaloids contained in this plant were historically used as a dye and for medicinal purposes and have recently drawn attention due to their antitumor activity. The use of TLC over high-performance liquid chromatography (HPLC) is a convenient way to reduce the amount of material, equipment and time needed for the analysis, and coupling of TLC with SERS provides vibrational information on each compound in the mixture. HPLC analyses with diode array detection were also carried out as a test of our technique, to ascertain the composition of Syrian rue extract and validate the results obtained from TLC-SERS investigations. In addition, Syrian rue extract and its commercial alkaloid components were characterized by SERS and normal Raman spectroscopy here for the first time, in order to provide valuable reference data to be used for identification purposes. Copyright © 2012 John Wiley & Sons, Ltd.