A method to identify mutations of virus proteins by using protein mass mapping is described. Comparative mass mapping was applied to a structural protein of the human rhinovirus Cys1199 --> Tyr mutant and to genetically engineered mutants of tobacco mosaic virus. The information generated from this approach can rapidly identify the peptide or protein containing the mutation and, in cases when nucleic acid sequencing is required, significantly narrows the region of the genome that must be sequenced. High-resolution matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and tandem mass spectrometry were used to identify amino acid substitutions. This method provides valuable information for those analyzing viral variants and, in some cases, offers a rapid and accurate alternative to nucleotide sequencing.