Glyphosate is a non-selective broad-spectrum herbicide widely used for weed control. It is currently one of the most important and widely sold herbicides in the world. Due to uncontrolled use and poor waste disposal, this herbicide has the potential to reach aquatic ecosystems, either surface water or groundwater, such as well water. The objective of this study was to show the degree of contamination of well water in 7 different agricultural areas of the Rabat-Sale-Kenitra region in Morocco. This is a prospective study carried out on 82 samples collected. The determination of glyphosate concentrations in these well waters was performed using the Enzyme-Linked Immunosorbent Assay (ELISA) technique. The results reveal that 83% of the samples collected showed traces of glyphosate (>0.075 µg/l) with concentrations ranging from 0.075 µg/l to 3.828 µg/l. The highest glyphosate concentrations were observed in the agricultural area of Kenitra, with a concentration of 3.828 µg/l. This study is one of the first conducted on glyphosate contamination of well water in Morocco. These results demonstrated glyphosate contamination of collected well water and the requirement to implement concrete sanitary measures (control, awareness campaigns...) to better manage the use of glyphosate and limit water contamination, the human health risks and environmental impact of this herbicide.