A method for the rapid limited enzymatic cleavage of PVDF membrane-immobilized proteins is described. This method allows the fast characterization of PVDF blotted proteins by peptide mass fingerprinting (Henzel, W. J., Billeci, T M., Stults, J. T, Wong, S. C., Grimley, C., Wantanabe, C., Proc. Natl. Acad. Sci. USA 1993, 90, 5011-5015), LC-MS/MS, or N-terminal sequencing and has been demonstrated on a range of proteins using a full complement of proteolytic enzymes. This technique allows the generation of proteolytic fragments between 5 and 60 min (depending on the enzyme employed), which is significantly faster than previously reported on-membrane digestion methods. To date, this on-membrane rapid digestion protocol has aided in the identification and confirmation of mutation sites in over 200 recombinant proteins.