Thioesters are ubiquitous functional groups in both chemistry and biology owing to their unique chemical properties. Thioester bonds are less stable than ester or amide bonds, but they are relatively stable in physiological environment. My main focus is the chemical biology of thioesters in this thesis. In the first part, I demonstrated the development of several chemical tools to study the protein S-palmitoylation, a biological original thioester. These chemical tools including a second-generation fluorescence-based turn-on depalmitoylation probe DPP-5 (Chapter 2) and mitochondrial-targeted APT inhibitor mitoFP (Chapter 3) to probe S-palmitoylation “eraser” activity in the live cells, as well as a fluorescence-based turn-on palmitoylation probes for high-throughput screening of S-palmitoylation “writer” inhibitors (Chapter 4). Furthermore, I demonstrated how these chemical tools could be applied to biological research by showing the discovery of a mitochondrial S-depalmitoylase, ABHD10, and how S-depalmitoylation regulates mitochondrial redox homeostasis by one of the ABHD10's substrates, PRDX5. In the second part, I first explored the utility of highly reactive thioester in the biomolecule labeling. Then I employed a bio-orthogonal ester-esterase technology developed by our lab towards the creation of an RNA labeling method via the unique ester-masked enol ester acylating reagents (Chapter 5). It is my hope that chemical tools described in this thesis will facilitate more discoveries in the protein S-palmitoyaltion research, which in turn fuels advancements in pharmacological tools to understand and treat human diseases. I also expect the ester masked thioester system could be further optimized for biomolecule proximity labeling purposes, and also provide insights to the development of novel approaches to mask highly reactive species.