Enzyme inhibition plays a crucial role in understanding enzyme kinetics, drug discovery, and biochemical regulation. Determining the inhibitory effects of various compounds on enzymatic activity provides valuable insights into their potential therapeutic or toxicological properties. The Ellman method, based on the colorimetric detection of thiocholine produced by the hydrolysis of acetylthiocholine, is one of the most widely used and reliable spectrophotometric techniques for assessing cholinesterase enzyme activity. In this study, the inhibitory effects of selected compounds on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) were evaluated using the Ellman assay. The method involves the reaction of the thiol group of thiocholine with 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) to produce a yellow-colored 5-thio-2-nitrobenzoate anion, measurable at 412nm. Kinetic analyses were performed to determine inhibition types and constants (Ki values). The obtained data demonstrated that the tested compounds exhibited varying degrees of inhibition, suggesting their potential use as enzyme modulators or drug candidates. The study confirms the efficiency, sensitivity, and reproducibility of the Ellman method for enzyme inhibition analysis, emphasizing its importance in biochemical and pharmacological research.