Acetylthiocholine iodide is primarily used in biochemical research as a substrate for cholinesterase enzymes. It plays a crucial role in studying enzyme activity, particularly in assays that measure the function of acetylcholinesterase and butyrylcholinesterase. These enzymes are essential in the nervous system, and their inhibition is a key mechanism in certain pesticides and nerve agents. By using this chemical, researchers can evaluate the effectiveness of inhibitors and develop potential treatments for conditions like Alzheimer’s disease, where cholinesterase activity is disrupted. Additionally, it is employed in histochemical staining to localize cholinesterase activity in tissues, aiding in the study of neurological structures and their functions.