Glyphosate is a broad-spectrum herbicide widely used worldwide. Indeed, it is the herbicide most applied to all Mexican crops. Due to the overuse and poor disposal of the waste, this herbicide can reach the aquatic environments such as groundwater and surface water. Thus, there is a clear need to implement monitoring and surveillance programs for evaluating and controlling the exposure to this herbicide in rural populations. The goal of this study was to quantify the presence of glyphosate in different water bodies (groundwater, surface and drinking water) as well as to identify the uses and managements of water resources by rural communities to evaluate the potential human exposure to glyphosate in the Tenampulco region of the Mexican state of Puebla. Measurements were performed by a rapid and cost-effective ELISA-based method in groundwater and surface water from various sampling sites of the Tenampulco region. Glyphosate was detected in all groundwater samples to be below the maximum limit for glyphosate in water in Mexico. Nevertheless, these results indicate an exposure of glyphosate in these agricultural communities and the need to establish a monitoring program.