One of the most widely used herbicides in the world, glyphosate, has contaminated waterways, posing a threat to both human health and the environment. The effectiveness of Lemna sp. (duckweed) in removing glyphosate (GLY) from contaminated aquatic medium in a lab setting is examined in this study. Plants were taken from the Diyala River in Iraq and placed on Schenk and Hildebrandt (SH) medium. After being treated with varying concentrations of glyphosate (25–150 mgl−1), growth parameters, chlorophyll contents (a, b total), and the effectiveness of glyphosate removal were measured after seven and fifteen days. Using High-Performance Liquid Chromatography (HPLC) to Determine Residual Glyphosate. The glyphosate residue levels were ascertained using this technique. According to the results, after 15 days, Lemna sp. had a maximum clearance effectiveness of 87% at 75 mg/L. Chlorophyll content increased dramatically at low concentrations (50–75 mg L-1), indicating metabolic adaptability and potential glyphosate phosphorus utilization. Lemna sp. exhibits faster recovery and greater resistance to glyphosate in comparison to other phytoremediation plants, such as Cicer arietinum and Eichhornia crassipes. According to these results, duckweed may be an effective, affordable, and eco-friendly substitute for glyphosate-polluted aquatic systems.