The use of statolith chemistry to trace migration pathways and distinguish populations of cephalopods is based on the assumption that the elemental composition of statoliths is influenced by physicochemical properties of the ambient environment. However, such influences have not been investigated experimentally up until now. This study presents the first microchemical analyses of cephalopod statoliths obtained from laboratory experiments under different controlled temperature and salinity conditions. Our results show that statolith chemical composition is strongly related to both salinity and temperature in ambient waters. The Ba/Ca ratio is negatively related to temperature and shows no relation to salinity. The I/Ca ratio is positively related to temperature and negatively to salinity. No Sr/Ca relation was found to either salinity or temperature, suggesting that the well-established proxy strontium is not as useful in cephalopod statoliths as in other biomineralized aragonites. Microanalysis of trace elements, however, shows an enormous potential for field studies on distribution, migration and stock separation of cephalopods. Furthermore, Synchrotron X-ray Fluorescence Analysis is introduced as a promising novel method for statolith analysis, providing a spatial resolution of typically 10–15 μm combined with detection limits down to 0.5 ppm.