Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was optimized to discriminate between wild-type and ampicillin-resistant Escherichia coli. Only ampicillin-resistant E. coli displayed an m/z approximately 29,000 peak, which was confirmed as beta-lactamase by in-gel digestion followed by peptide mass fingerprinting. Rapid MALDI-TOF MS detection of antibiotic-resistance could fulfill an important clinical need, providing critical phenotypic information beyond genus-species identification.