The Western diet of high fructose and high fat has led to a rise in metabolic diseases such as obesity and diabetes due to its effect on metabolism and nutrient availability throughout the body. Short-chain fatty acids (SCFAs), such as butyric, acetic, and propionic acids, are made by microbiota in the gut from ingested nutrients. SCFAs have a role in promoting healthy immune and gut function, as well as maintaining overall intestinal homeostasis. Enterocytes that line the gut lumen use SCFAs as an important energy source, as well as converting fructose into glucose. However, a high fructose intake may overwhelm the process, causing unconverted fructose to reach the colon where the microbiota converts the fructose into SCFA’s, feeding liver lipogenesis. Due to the low concentration of SCFAs throughout the body and their volatile nature, these SCFAs may be difficult to measure. Gas Chromatography and Mass Spectrometry (GCMS) have been the preferred methods for analyzing SCFAs for its high sensitivity. By studying biological samples from mice fed a diet of high fructose and fats, we hope to measure changes in the metabolic production of SCFAs from C¹³-enriched fructose through isotope tracing. Measuring labeled and unlabeled fatty acids within the biological samples of mice fed with controlled diets shows that a high-fat diet alters SCFA production by the microbiota, leading to the negative effects of consuming a Western diet.