Thioesters are considered to be “energy-rich” functional groups that are susceptible to attack by thiolate and amine nucleophiles while remaining hydrolytically stable at neutral pH, which enables thioester chemistry to take place in an aqueous medium. Thus, the inherent reactivity of thioesters enables their fundamental roles in biology and unique applications in chemical synthesis. Here, we investigate the reactivity of thioesters that mimic acyl-coenzyme A (CoA) species and S-acylcysteine modifications as well as aryl thioesters applied in chemical protein synthesis by native chemical ligation (NCL). We developed a fluorogenic assay format for the direct and continuous investigation of the rate of reaction between thioesters and nucleophiles (hydroxide, thiolate, and amines) under various conditions and were able to recapitulate previously reported reactivity of thioesters. Further, chromatography-based analyses of acetyl- and succinyl-CoA mimics revealed striking differences in their ability to acylate lysine side chains, providing insight into nonenzymatic protein acylation. Finally, we investigated key aspects of native chemical ligation reaction conditions. Our data revealed a profound effect of the tris-(2-carboxyethyl)phosphine (TCEP) commonly used in systems where thiol-thioester exchange occurs, including a potentially harmful hydrolysis side reaction. These data provide insight into the potential optimization of native chemical ligation chemistry.