An unexpected chromatographic behavior of fat globules has led to a simple HPLC assay for glycerin in the complex matrix of fat emulsions intended for intravenous injection. When a combination of a Hamilton PRP-1 (Polymer Reverse-phase) Column, a Biorad Microguard Carbo C Guard Column, and a Biorad HPLC Carbohydrate Analysis Column connected in series in the order listed was used for the separation of glycerin, with water as the mobile phase, the fat globules eluted unretained. The glycerin, which eluted by aqueous size exclusion, was detected and quantitated using a refractive index detector.