Karst springs are one of the most exploited drinking water resources in the world. Isotopic and chemical analyses on these waters reveal essential insights into their hydrodynamics. Valseriana (Northern Italy) is a valley within the Italian Pre-Alps, characterized by high water availability. The main reasons are a bedrock consisting of very permeable carbonate formations and a mean annual precipitation above 1800 mm/y. In this context, the Valseriana spring catchments have become strategically important for the water exploitation and the domestic supply of Bergamo and the neighboring municipalities. This study general aim is to expand the knowledge on the two main karst systems (Nossana and Ponte del Costone), which together include twenty-three major and minor springs, by defining a state-of-the-art that will be useful in future decades to preserve this crucial resource. Specific objectives include i) verifying the applicability of the 3H/3He dating method in karst environments, ii) defining groups of springs according to their chemical and isotopic characteristics for possible safeguard actions, and iii) establishing a conceptual model of the internal dynamics of the two spring systems. A chemical and isotopic sampling campaign was specifically conducted between May 2018 and July 2019. A total of 34 water points were sampled, including natural springs, rivers, wells, karst caves, and nearby mines. Water sampling for 3H/3He isotopic analyses was proved possible through careful selection of the sampling points and the insertion of the pump tubing in the spring fractures. A first classification of springs into uniform hydrochemical groups was obtained by applying the hierarchical cluster analysis technique on major ions contents. These outputs were combined with information from the 3H/3He dating analyses and resulted in three different classes of springs. The three classes showed a clustering for elevations: at high, medium, and valley bottom elevations, respectively. Finally, the elaborations simplified the two karst systems by a hierarchical flow system model, dynamically controlled by different karst network development and infiltration water.