Acetylcholinesterase inhibitors (AChEI) are currently still the best available pharmacotherapy for Alzheimer patients. Successful screening for new AChEI relies on effective and fast assays. Two colorimetric screening assays frequently used to search for new AChEI, namely a thin layer chromatography (TLC) assay with Fast Blue B salt as reagent and a 96-well plate assay based on Ellman's method, were compared. For the majority (83%) of the 138 test compounds of natural and synthetic origin, the results obtained with the two assays converged and both screening assays were considered suitable for the generation of new hits. Fifteen percent of investigated compounds were classified as active with the microplate assay but were shown to be inactive by TLC and about 2% were measured active by TLC but showed to be inactive with the microplate assay. These divergences were not due to the main differences between the experimental protocols of the two screening assays, namely the different colorimetric methods and pre-incubation of test compounds with acetylcholinesterase (AChE). They might be explained by the interaction of either AChE or test compounds with the silica of the TLC plates, resulting in an altered affinity of the enzyme for the compounds.