Abstract The chromogenic reaction of cholesterol with iron-sulfuric acid-acetic acid has been modified to increase its sensitivity, precision, and stability. This has been done by carrying out the reaction isothermally (at room temperature), at the same time avoiding exposure to fluorescent light or sunlight. Optimal conditions for maximal yield of chromogen have been established, as a result of which the sensitivity is increased greatly over previous methods. The modified chromogenic reaction is applied to fractions obtained by a modified Abell extraction schema which allows the accurate and precise measurement of the total cholesterol of serum using 0.1-ml. samples.