Trypsin was immobilized by covalent binding to glutaraldehyde-activated silica with and without a spacer arm; 1,6-diaminohexane and polyethyleneglycol as well. The addition of polyethyleneglycol (PEG) to the immobilization media increased the activity of immobilized trypsin in organic solvents, whilst free trypsin activity disappeared under the same conditions. Thermal, pH, storage, and operational stabilities of the free and immobilized enzyme were found to be better than the free enzyme. Furthermore, use of immobilized enzyme for protein fragmentation was achieved by solid-phase, on-line, protein digestion in organic solvents. Reaction times were reduced to a few minutes and the sample handling was minimized.