High-throughput and efficient analytical approaches for bioassay-guided fractionation and chemical profile of crude extracts are still a challenge. Nowadays researches are focusing on advanced hyphenated techniques that enable the direct identification of compounds with the desire biological effect like on-line high-performance liquid chromatography (HPLC) with in vitro biochemical assay. The use of immobilized enzyme reactors (IMER) as an on-line post-chromatographic biochemical assay is a promising alternative. Immobilization procedure can increase enzymatic stability against heat, organic solvent and pH without too much loss of catalytic activity allowing the direct coupling to a liquid chromatographic separation system using a sampling valve as interface. Our group proposed an on-line comprehensive in vitro bioassay using an immobilized capillary acetylcholinesterase reactor (ICER-AChEeel) coupled to mass spectrometry detection. This, proved to be a powerful method for the fast screening of inhibitors in natural products crude extracts. Two well established acetylcholinesterase inhibitors, galantamine and donepezil, were used as proof of concept. The two-dimensional system described in Fig. 1. allowed recognition of both compounds as acetylcholinesterase inhibitors by direct monitoring acetylcholinesterase hydrolysis product choline’s (Ch) precursor ion m/z 104.0 ([M+H]+) and its fragment m/z 60.0 ([M+H-C2H3OH]+)[1]. The combination of the analytical separation with on-line AChE enzymatic assay coupled to mass spectrometry increases the likelihood of identifying individual inhibitors compared to conventional assays reducing considerably the time required to assign a biology activity to a chemical structure.