Artificial sweeteners are sugar substitutes added to foods, beverages, and various pharmaceutical and personal hygiene products. Sucralose and saccharin – two highly used artificial sweeteners – are not metabolized by humans and are poorly removed during wastewater treatment, resulting in their widespread release to environmental waterbodies. Constant discharge, increasing usage, and environmental persistence of artificial sweeteners yield worsening chronic exposure conditions for aquatic organisms. Artificial sweeteners are not acutely toxic; however, they represent a potentially significant threat to aquatic life because of their ubiquity and understudied chronic effects. Stressors, including contaminant exposure, often cause rapid perturbations in metabolite abundance. Thus, metabolomics – the comprehensive study of an organism’s metabolites at a specific time – is a sensitive approach to detect sublethal health effects. We exposed adult rainbow trout (Oncorhynchus mykiss) to either 20 or 200 µg/L aqueous sucralose or saccharin for 28 days. Sample analysis is currently ongoing to identify biomarkers (e.g., proteins and polar metabolites) in trout blood plasma and tissues using liquid chromatography and tandem mass spectrometry. We will then apply bioinformatics tools to map identified biomarkers to their biological pathways and present our resulting preliminary findings on the environmental health threat of artificial sweeteners.