Due to karst hydrological systems’ heterogeneity and complexity, its investigation is quite demanding. Use of artificial tracers on karst terrains provides an insight into the functioning of short groundwater channels, while environmental tracers enable the analysis of different karst subsystems. Stable isotopes of water are perfect environmental tracers and they serve as water molecule tracers through the hydrological cycle. There are three stable isotopes of oxygen (16O, 17O and 18O) and two stable isotopes of hydrogen (1H and 2H), that form nine possible configurations of stable water molecules. Stable isotope composition of water is expressed in terms of δ-values (δ2H, δ18O) that represent a relative difference between ratios of less abundant and predominant isotopes in a sample and in an internationally accepted standard: δ (‰)=(Rsample/Rstandard)-1. Construction of Local Meteoric Water Line (LMWL, the linear regression line for δ2H and δ18O in precipitation of certain area) and its comparison to the Global Meteoric Water Line (GMWL, δ2H=8∙δ18O+10 ‰), as well as an analysis of deuterium excess (d-excess= δ2H-8∙δ18O) are essential parts of one of the main isotope hydrology applications that is identification of a groundwater origin. The differences in slope and intercept between the GMWL and the LMWL are the consequences of climate conditions and geographical parameter variations. Stable isotopes of water have been applied in investigations of karst aquifers to understand recharge processes, reservoir mixings, to determine mean recharge area elevations, for identification of water sources in karst springs, etc.