Acetylthiocholine chloride is primarily used as a substrate in enzymatic assays, particularly for measuring the activity of cholinesterase enzymes. It plays a critical role in research and diagnostics to evaluate the function of acetylcholinesterase and butyrylcholinesterase, which are essential for nerve signal transmission. This compound is also utilized in toxicology studies to detect the presence of cholinesterase inhibitors, such as organophosphate pesticides and nerve agents, by monitoring changes in enzyme activity. Additionally, it finds application in biochemical research to study enzyme kinetics and mechanisms of cholinesterase inhibition.