A procedure for determining the esterase content of biologic tissues and fluids has been presented. Fatty acid esters of 2-naphthol are employed as substrates. The 2-naphthol liberated by hydrolysis is coupled with a diazonium salt to form an azo dye, the concentration of which is estimated photometrically. Distinctive features of this method are (1) the use of Brij 35 to prepare nonopalescent solutions of substrate,(2) the adjustment of pH to ensure complete formation of the azo dye, and (3) the solubilizing of the azo dye by adding sodium hydroxide. This assay procedure is sensitive to very small amounts of esterase, and the stability of its color is excellent if proper precautions are taken.