A microchip reactor coated with a gold nanoparticle network entrapping trypsin was designed for the efficient on-line proteolysis of low level proteins and complex extracts originating from mouse macrophages. The nanostructured surface coating was assembled via a layer-bylayer electrostatic binding of poly(diallyldimethylammonium chloride) and gold nanoparticles. The assembly process was monitored by UV-visible spectroscopy, atomic force microscopy, and quartz crystal microbalance. The controlled adsorption of trypsin was theoretically studied on the basis of the Langmuir isotherm model, and the fitted !max and K values were estimated to be 1.2 X 10-7 mol/m2 and 4.1 X 105 M-1, respectively. An enzymatic kinetics assay confirmed that trypsin, which was entrapped in the biocompatible gold nanoparticle network with a high loading capacity, preserved its bioactivity. The maximum proteolytic rate of the adsorbed trypsin was 400 mM/(min.µg). Trace amounts of proteins down to femtomole per analysis were digested using the microchip reactor, and the resulting tryptic products were identified by MALDI-TOF MS/MS. The protein mixtures extracted from the mouse macrophages were efficiently identified by online digestion and LC-ESI-MS/MS analysis.