A proteomics approach is reported for the rapid recognition of genetically modified Escherichia coli bacteria. The approach targets a class of proteins required for genetic manipulation of bacteria with plasmids and alleviates the need to construct extensive libraries of toxins and other predicted payload proteins. Detection was performed using MALDI-TOF MS to monitor peptide products after an on-probe enzymatic digestion. Digestion products were identified by searching their postsource decay spectra using MASCOT. A 5 min digestion time was required to observe peptide products from the genetic insert as well as the host bacterium. This proteomics approach enables rapid detection of genetic manipulation along with information about the host organism, both of which have forensic applications.