The aim of the here presented study was to combine high performance liquid chromatography with plate reader technology in order to overcome certain drawbacks of integrated online systems as well as offline plate reader approaches. The described method combines an "at-line" enzyme assay for the simultaneous bioactivity determination with parallel QTOF MS data acquisition for analyte identification. All biochemical reagents are added in an online mode directly to the column effluent (postcolumn addition/mixing), and the complete screening assay mixture is subsequently microfractionated into a 1536 well plate. The screening of a natural extract fortified with two well-known Protein Kinase A inhibitors and the identification of an inhibitor in a natural extract showed the applicability of the approach to detect bioactive compounds in low concentrations in a complex mixture. The described mode of operation utilizes today's plate reader technology to its full capacity and directly hyphenates it to a high resolution separation technique which has not been shown before. Furthermore, it allows coupling of a microbore HPLC with a biochemical screening assay without compromising resolution and overcomes problems associated with the 1536 well format. © 2009 American Chemical Society.