Accurate diagnosis of Clostridium difficile infection (CDI) is important not only for patient care but also for epidemiology and disease research. As it is not possible clinically to reliably differentiate CDI from other causes of health care–associated diarrhea, the laboratory confirmation of CDI is essential. Rapid commercial assays, including nucleic acid amplification tests and immunoassays for C difficile toxin and glutamate dehydrogenase, have largely superseded the use of older assays. Although assays that detect the presence of free C difficile toxin in feces are less frequently positive than tests for organism, they are preferable for the detection of CDI.