The use of immobilized capillary enzyme reactors (ICERs) for on-line ligand screening has been adopted as a new technique of high throughput screening (HTS). In this work the selected target was the enzyme acetylcholinesterase (AChE) and the AChE-ICERs produced were used in a LC-tandem ion-trap mass spectrometer (IT-MS/MS). The activity and kinetic parameters were evaluated monitoring the choline's precursor ion (M+H)⁺ 104.0 m/z and its ion fragment (C₂H₃OH)-(M+H)<sup+ 60.0 m/z . The assay method was validated using the reference AChE inhibitors: tacrine and galanthamine. Two new ligands, out of a library of 17 coumarin derivatives, were identified and the half maximal inhibitory concentration (IC₅₀), inhibition constant (K_i), and the inhibition mechanism determined. A coumarin derivative with IC₅₀ similar to tacrine was highlighted.