Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a new technique that can be used to determine the molecular composition of polysorbate emulsifiers, which are commonly used as food additives. This is the first study to offer such a detailed examination of these heterogeneous compounds. MALDI-TOF MS is a powerful tool that can provide a polysorbate mass profile in less than two minutes. 2',4',6'-Trihydroxyacetophenone monohydrate was chosen to be an ideal matrix, as it easily facilitated desorption and ionization, provided good resolution, and allowed for fast and simple preparation of the sample. By addition of aqueous 0.01 M potassium chloride, species were resolved exclusively as potassium adducts in the positive ion mode. MALDI-TOF MS analysis before and after saponification indicated the presence of unbound ethylene oxide polymers, as well as free and esterified sorbitan- and sorbide-based species. Some evidence for the presence of disorbitan-based species was provided. Also illustrated were the polydispersity of the oxyethylene chains, the degree of esterification, and the identity of esterified fatty acids.