Water security requires that sufficient quantities of water be available at critical times. This is particularly challenging for high-intensity urban and agricultural settings. In underdeveloped nations, streamflow is commonly the preferred water source, as it is readily available and delivered cost-free to users. Yet, the sources of these critical streamflows are often unknown. This issue is salient in the Northern Andes, where basic knowledge of controlling factors for the quantity, quality, and timing of runoff is lacking. High-elevation headwaters are the primary catchment areas in the Northern Andes, but the extent of water providing to municipalities in the Northern Andes is unknown. In this study, the contribution of water derived from the upper watershed to the streamflow in the Tulúa River which supplies water to 200,000 people in the city of Tulúa was quantified. The river runs 72 km through urban, agricultural, and industrial land use in the Central Cordillera of the Colombian Andes. We collected 32 and 34 water samples in August and November, respectively. The water samples were representative of high-elevation headwaters runoff, shallow groundwater discharge, and streamflow throughout the watershed. Samples were analyzed for dissolved constituents and stable isotopes. The dissolved constituents were used in mass-balance mixing models to identify the source of streamflow in the lower watershed of the Tulúa River, where it the river supports a large municipality. Results indicate that approximately 50% surface runoff largely originates as high-elevation headwater runoff, including high-elevation settings where páramos dominate the land cover. These findings underscore the need for source-water protection efforts in the upper watershed, including the páramos. This project serves as a model for other páramo derived watersheds, where source-water protection is a critical challenge.