We evaluated a new method for measurement of acetaminophen. From acetaminophen, acetate and p-aminophenol are enzymically produced, and the latter product is determined colorimetrically by reaction with o-cresol and ammoniacal copper sulfate. The method is rapid and precise, and results compare well with those by spectrophotometric and liquid-chromatographic techniques. Major metabolites of acetaminophen, some commonly used drugs, or phenolic substances that may be present in patients with liver or renal disease do not interfere.