Lakes provide an important water resource for agricultural and industrial purposes, and form a critical habitat for plants and animals. Understanding the hydrological functioning of lakes is fundamental to understand hydrological feedbacks related to changing environmental conditions in the context of current climate change and also of increasing importance for environmental policy. Stable isotope ratios of water (2H/1H, 18O/16O) are widely used in lake hydrology studies, providing information on the hydrological balance of lake systems. However, in cases where the system is under-defined, a full quantitative assessment of the lake’s hydrological balance is challenging. Recent investigations have demonstrated that the additional analysis of the rare oxygen-17 isotope can provide additional quantitative information about the hydrological and environmental conditions during lake evaporation. The HydrO-17 Project will explore the potential of the triple oxygen isotope analysis (18O, 17O, 16O), expressed as 17O-excess, for quantitative hydrological balancing of lakes by examining the seasonal isotope evolution of hydrologically different lake systems in the Andalusian lowlands, Southern Spain, where agricultural overexploitation of groundwater aquifers over the last decades led to a severe water deficit. Current climate change will further intensify these problems. The results of the project will contribute to the understanding of the hydrological functioning of lake systems in Andalusia, with implications for local water management strategies. The approach has potential application to lakes in other regions in the world. The outcomes will also help to improve the interpretation of paleoclimate and paleoenvironmental information obtained from paleo-lake sediment archives.