A continuous spectrophotometric assay for pantetheinase determination using S-pantetheine-3-pyruvate as substrate is described. The enzymatic hydrolysis of this new substrate leads to the formation of S-cysteamine-3-pyruvate, which cyclizes in a non-rate-limiting step to give 2H-1,4-thiazin-5,6-dihydro-3-carboxylic acid (aminoethylcysteine ketimine), a compound exhibiting a strong absorption at 296 nm. The assay is optimized with respect to pH, buffer, and substrate concentration. Prereduction of the enzyme and some properties of the reaction are also studied. The assay is simple, rapid, very sensitive, and specific.