The aim of this study was to screen for acetylcholinesterase (AChE) inhibitors from a large chemical library of commercially available compounds. For this purpose, the Ellmans reaction based assay was miniaturized into 384-well plate format, and two modifications of the kinetic protocol were studied with the aim of developing a rapid screening platform that could ensure high efficiency in finding true hits. It was proven that when starting the kinetic reaction by addition of the substrate, better assay performance was achieved and more practical benefits obtained. Using the optimized automated protocol, a chemical library of 56,320 compounds was screened. A total of 350 positive hits were identified and their IC50 calculated. Three highly active compounds were identified with IC50 values close or even lower to physostigmine ( < 0.1 μM). The activity towards butyrylcholinesterase (BChE) of these three most active hits was also evaluated. The most active hit (IC50(AChE) = 0.019 μM), was identified as a new inhibitor, belonging to ChemDiv chemical library: (N-[3-(3,5-dimethyl-1-piperidinyl)propyl]-5-ethyl-2-methyl-8-oxo-thieno[2,3:4,5]pyrrolo[1,2-d] [1,2,4] triazine- 7(8H)-acetamid), with no other biological activities reported until now. The interactions of this hit with both cholinesterases were further analyzed using computational docking studies. To our knowledge, this is the largest published screening campaign of commercially available compounds that has focused on finding new AChE inhibitors. The miniaturized 384-well plate format of the Ellmans method was proven to be robust and to perform reliably.