Cysteine residues are highly versatile amino acids that control a variety of protein properties. Due to their critical roles in dictating protein structure and function, cysteines directly contribute to the maintenance of protein homeostasis (proteostasis) and healthy cellular function. The unique reactivity of cysteine residues allows them to undergo a number of modifications, including oxidative marks and alkylation by electrophilic small molecules, which can both promote and inhibit the function of cysteine-containing proteins. This feature has been extensively leveraged for the development of covalent therapeutics. Consequently, cysteine chemoproteomics has emerged as a powerful tool for identifying cysteines that are amenable to oxidative and covalent modification proteome-wide, offering the opportunity to identify potentially functional and “druggable” residues. However, these chemoproteomic platforms do not inherently read out the functional impact of a cysteine modification event, and thus delineating the functional impact of cysteine compound labeling or oxidative modification is a central challenge in the field. Furthermore, the global impact of covalent molecules on cellular proteostasis processes has not previously been described, and the extent to which covalent-induced proteome rewiring can confound the interpretation of specific structure activity relationships (SAR) is an open question. Here, I present my thesis work, which describes the development and application of chemoproteomic and biochemical techniques to characterize the impact of covalent small molecules on proteostasis processes and to delineate the cysteine-centric regulation of cell stress response mechanisms. First (Chapter 2), I review methods for identifying small molecule modulators of RNA-binding proteins (RBPs), a class of proteins that has been classically considered “undruggable,” and describe how chemoproteomics approaches can be utilized to bridge this druggability gap for RBPs. Included in this discussion is an analysis of ligandable amino acids within RBPs that have been identified by previous chemoproteomics campaigns. Next (Chapter 3), I describe our efforts in illuminating the generalizable proteome rewiring that occurs in response to covalent small molecules. We start by characterizing the covalent-induced depletion of SARS-CoV-2 nonstructural protein 14 (nsp14), which is promoted by covalent labeling of both nsp14 cysteines and cysteines within host protein disulfide isomerases, leading to protein aggregation and depletion. We then describe the global effects imparted by covalent small molecules, including increased protein ubiquitination, increased proteasome activity, aggresome formation, and stress granule (SG) induction. Having described the generalizability of SG induction by cysteine-reactive small molecules, we finally (Chapter 4) develop and employ a suite of innovative chemoproteomic platforms to identify cysteines that are modified, either by covalent small molecules or oxidative modifications, to modulate protein phase separation. We establish a high-throughput SG imaging assay to describe the kinetics of covalent and oxidation induced SGs, utilize a structurally matched covalent atropisomer pair to identify cysteines preferentially engaged by SG-inducing compounds, and develop a SG-localized dual enrichment redox proteomics platform to interrogate the redox sensitivity of SG cysteines. We identify cysteines in the fragile X-related protein family (FXR1_C77 and FXR2_C282) that impact the proteins’ ability to phase separate into SGs, representing functionally important cysteines that may act as nodes of SG regulation. Collectively, my thesis work lays the foundation for understanding the cysteine-centric regulation of the cell stress response, particularly as it relates to proteostasis manipulation and stress granule dynamics. I am optimistic that this work can provide insight into cellular processes that are manipulated by covalent compounds, potentially complicating interpretation of specific SAR, and also illuminate functionally important cysteine residues that can be used as potential starting points for interrogating and manipulating the cell stress response.