A novel approach to rapid bacterial detection using an isotopically labeled (15)N bacteriophage and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) is introduced. Current phage amplification detection (PAD) via mass spectrometric analysis is limited because host bacteria must be inoculated with low phage titers in such a way that initial infecting phage concentrations must be below the detection limit of the instrument, thus lengthening incubation times. Additionally, PAD techniques cannot distinguish inoculate input phage from output phage which can increase the possibility of false positive results. Here, we report a rapid and accurate PAD approach for identification of Staphylococcus aureus via detection of bacteriophage capsid proteins. This approach uses both a wild-type (14)N and a (15)N-isotopically labeled S. aureus-specific bacteriophage. High (15)N phage titers, above our instrument's detection limits, were used to inoculate S. aureus. MALDI-TOF MS detection of the (14)N progeny capsid proteins in the phage-amplified culture indicated the presence of the host bacteria. Successful phage amplification was observed after 90 min of incubation. The amplification was observed by both MALDI-TOF MS analysis and by standard plaque assay measurements. This method overcomes current limitations by improving analysis times while increasing selectivity when compared to previously reported PAD methodologies.