A microbiological assay has been developed using the techniques of assay for antibiotics. Metal cylinders were placed on plates prepared by overlaying a thin layer of seed agar, inoculated with a test organism, on a base of nutrient agar. Appropriate dilutions of a standard glycerol solution and of the glycerol preparations being tested were added to the proper cylinders. The plates were incubated and the zone sizes around the cylinders measured. The potency of the preparations was determined by comparing the average zone size of the unknown preparations with that of the standard. The method proved to be simple to perform, and in the absence of interfering substances, yielded essentially the same results as chemical methods. A number of carbohydrates—glucose, laevulose, maltose, mannitol, mannose, saccharose, and trehalose—interfered with assay results so that the method cannot be used for glycerol determinations in mixtures where they are present. No interference, however, was noted with d-arabitol and erythritol, which were found to interfere with the chemical assay.