The search for pharmacological treatments for Alzheimer’s disease has long centered around cholinesterase enzymes. While cholinesterase inhibitors may not directly prevent, slow down, or stop the progression of the disease, they remain a focus of research due to their potential in developing multifunctional compounds (multitarget directed ligands, MTDLs) that can target multiple pathways. In the framework of developing new compounds endowed with anticholinesterase activity, this chapter is dedicated to outlining in vitro methods for evaluating and understanding the mode of action of compounds with anticholinesterase activity. It will cover experimental protocols for assessing inhibitory activity using Ellman’s method, as well as the mechanism of inhibition for both reversible and pseudo-irreversible inhibitors. To ensure the relevance of these findings to human patients, the assays detailed in this chapter are optimized for use with human cholinesterases whenever possible.