Complex synthetic formulations based on polysorbates can be challenging to characterize. They may be composed of many similar products including those of the same molecular weight, which cannot be readily separated by separation science approaches. Carbon number variation and ethylene oxide distribution add to the complexity. The properties of these formulations will be dependent on the chemical structure and relative concentration of formulation components. Here we describe the use of two experimental approaches based on mass spectrometry to provide enhanced characterization of these formulations. The first utilizes an atmospheric pressure solids analysis probe to rapidly determine the percentage content of individual esters in a formulation. These are shown to be in good agreement with product specification sheets. In a second approach, mobility separation has been integrated into a MALDI-MS/MS experiment to categorize major, minor, and trace ingredients. Components of identical molecular mass in the polysorbate formulations have been separated by ion mobility and then fragmented for additional characterization. The rapidity and level of structural detail provided by these experiments offers a significant opportunity to develop practical screening methods for complex formulations.