Tracer methods have been widely used in many fields of environmental and natural sciences, and also in human health sciences. In particular, tracers are used in the study of karst hydrogeology, typically focusing on phenomena such as sinkholes, sinking rivers and large karst springs. It is known that tracers have been used since antiquity. The aim of tracer tests has been to investigate underground flow paths, transport processes and water–rock interactions, and to get an insight into the functioning of a karst aquifer. In karst hydrogeology, tracer methods are the most important investigation tools beside conventional hydrological methods. In early times, tracer methods were applied only to investigate underground flow-paths. Later they were also used to elucidate transport processes associated with water flow, and today they are often the basis, together with detailed hydrological information, of groundwater protection investigations and aquifer modelling. Many substances (spores, microspheres, bacteriophages, salt tracers, fluorescent dyes, radioactive substances) have been investigated for their properties and potential usage in environmental investigations, in particular the often unknown and inaccessible underground systems of karst areas. A great number of analytical techniques is available. This includes instrumentation for laboratory applications and direct online, on-site or in-situ field measurements. Modern instruments have a high capability for data acquisition, storage and transmission in short intervals, as a basis for quantitative evaluation and modelling. This enables research on the hydrological and hydrochemical dynamics of aquifers and their response to different natural or anthropogenic impacts.