Understanding protein higher order structure (HOS) is important because it is directly related to a protein’s function. These functions are wide-ranging, and misfolding of proteins can have significant adverse effects. Consequently, biophysical and biochemical approaches have been developed to study protein HOS in numerous different contexts. Covalent labeling (CL) coupled with mass spectrometry (MS) has become a powerful tool for probing protein HOS information in recent years, owing to its throughput, sensitivity, and sample efficiency which set it apart from traditional biophysical methods. This dissertation focuses primarily on the use of diethylpyrocarbonate (DEPC) as a CL-MS reagent. DEPC reacts with the side chains of multiple amino acids, allowing for coverage of 30% of the average protein’s sequence. Owing to its ease of use, DEPC has been used extensively by our group to study protein structure and interactions. DEPC readily reacts in solution to produce a single reaction product which is easily identified by tandem MS. The goal of this dissertation is to further the development and application of DEPC CL-MS tools to study protein structure and interactions. x The first part of this dissertation addresses the current experimental constraints placed on DEPC CL-MS studies. Through modeling the kinetics of the labeling reaction, bottom-up site mapping, molecular dynamics simulations, and ligand binding experiments, we show that proteins can accommodate more than one label at a time, allowing for improvement to the obtainable information from DEPC CL-MS experiments. Next, we apply DEPC to new systems: membrane proteins in live cells and nucleic acid binding to proteins. We show that DEPC can be applied in live cells in order to obtain information about membrane protein interactions and that DEPC can be used to probe protein-nucleic acid binding interactions. Lastly, we investigate the αα,ββ-unsaturated carbonyl (ABUC) scaffold as a CL-MS reagent specifically for ligand-directed labeling approaches. Similarly to DEPC, the ABUC reacts readily in solution and produces a single reaction product, but has a wider range of reactivity. This reagent provides better amino acid coverage compared to traditional ligand-directed approaches, expanding the current tools that are available.