Live mammalian cells are used extensively in both the laboratory and the clinic.Unfortunately, these cells are often fragile and cannot easily be manipulated or stored for long periods of time without a significant decrease in cell viability. We have developed a method for protecting mammalian cells that exploits the disaccharide trehalose, a naturally-occurring cellular protectant. In this method, we have synthesized cell-permeable trehalose analogues in which the hydrophilic hydroxyl groups of trehalose are masked as esters. Once inside the cell, these ester groups are cleaved by endogenous esterases and release free trehalose. We have previously demonstrated that two of our analogues deliver high concentrations of trehalose into mammalian cells. To better understand the esterase-catalyzed removal of esters from our trehalose analogues, we have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based esterase assay using porcine liver esterase (PLE), a mammalian esterase, as a model enzyme. We are currently evaluating the kinetics of PLE-catalyzed hydrolysis of our trehalose analogues. Results from these experiments will provide us with a better understanding of the production of free trehalose inside mammalian cells when using esterified trehalose analogues and will aid us in the design of more effective trehalose analogs.