Non-Alcoholic Fatty Liver Disease (NAFLD; also known as Hepatosteatosis) is a disease caused by the accumulation of lipids in liver cells that is not related to alcohol usage. In order to reduce the use of invasive liver biopsies for the diagnosis of NAFLD progression, various biomarkers for hepatosteatosis has been evaluated. Recent studies on searching possible metabolic biomarkers for NAFLD, it was shown alanine to pyruvate ratio is significantly increased in rats that developed NAFLD. The result closely correlates with the increased activities of alanine aminotransferase, which is an established biomarker for liver damage. Thus, the measurement of alanine/pyruvate ratio in serum or liver could be a promising marker to detect NAFLD. NAFLD detection kit determines the alanine to pyruvate ratio in two independent enzymatic reactions. The difference between these two measurements gives the actual amount of alanine and pyruvate present in the samples. This kit provides a simple, fast and high throughput adaptable assay to measure the ratio of alanine and pyruvate with in various biological samples in both colorimetric (O.D. 570 nm) with detection range of alanine and pyruvate 2-10 nmol and fluorometric mode (Ex/Em = 535/587nm) with detection range 0.2-1 nmol.