A high performance liquid chromatographic method is described for the assay of chloramphenicol acetyltransferase activity which detects the formation of both 3-acetoxy chloramphenicol and 1,3-diacetoxy chloramphenicol. In experiments performed at pH 6.8 only 3-acetoxy chloramphenicol was detected while in experiments performed at pH 7.8 both 3-acetoxy and 1,3-diacetoxy chloramphenicol were detected.