This diploma thesis deals with the determination and ecotoxicological assessment of artificial sweeteners. The diploma was focused on monitoring the most commonly used sweeteners, which includes acesulfam-K, cyclamate, saccharin and sucralose. These sweeteners were analyzed by liquid chromatography with mass spectrometry, using solid phase extraction for their concentration. The samples for their determination were taken from wastewater treatment plant in Modrice. Artificial sweeteners are synthetically produced substances, which is the main reason for their occurrence in waste water. Some of them are not efficiently removed from the wastewater treatment plant and penetrate into the environment. Analysis of the samples was found, that sucralose penetrates into the environment. The ecotoxicological effect of the sweeteners was evaluated via aquatic organisms, namely on the freshwater crustaceans Thamnocephalus platyurus and Daphnia magna, on the water plant Lemna minor, and the battery of tests was supplemented by a representative of the destructors Vibrio fischeri. For all sweeteners, LC20, EC20 and IC20 values were determined. For some sweeteners, LC50, EC50 and IC50 values were also determined. During testing, saccharin represented the highest toxicity.