Choline is an essential nutrient and plays a key role in many biological processes. Choline and its metabolites are needed for various physiological purposes, including: structural integrity of cell membranes, cell membrane signaling, cholinergic neurotransmission, and additionally, they are a major source of methyl groups that are needed for the biosynthesis of lipids and the regulation of metabolic pathways. Choline deficiency plays a role in liver disease, atherosclerosis, neurological disorders, and organ dysfunction. This colorimetric assay is based on choline oxidase-catalyzed oxidation of free choline, where the formed hydrogen peroxide is then catalyzed by peroxidase and reacts with 4-aminoantipyrine to form a colored product that can be read at 550nm. The intensity of the colored reaction product is directly proportional to the concentration of choline in the sample.