Being able to rapidly and sensitively detect specific enzymatic products is important when screening biological samples for enzymatic activity. We present a simple method for assaying protease activity in the presence of protease inhibitors (PIs) by measuring tryptic peptide accumulation on copolymer pMALDI target chips using a dual fluorescence/MALDI-TOF-MS readout. The small platform ofthe chip accommodates microliter amounts ofsample and allows for rapid protein digestion. Fluorescamine labeling of tryptic peptides is used to indicate the pro- teolytic activity and is shown to be an affordable, simple process, yielding a strong fluorescence signal with a low background. Subsequent MALDI- TOF-MS analysis, performed in the same sample well, or in a parallel well without adding fluorescamine, detects the specific tryptic pep- tides and provides confidence in the assay. The dual read-out method was applied to screen the inhibition activity of plant PIs, components of plant defense against herbivores and pathogens. Extracts of PIs from Solanum nigrum and trypsin were applied together to a pMALDI chip on which a suitable substrate was adsorbed. The fluorescence and MALDI-TOF-MS signal decrease were associated with the inhibitory effect of the PIs on trypsin. The developed platform can be modified to screen novel protease inhibitors, namely, those potentially useful for treating or preventing infection by viruses, including HIV and hepatitis C. © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.