This paper describes a method—using a combination of LC-MS/MS of selected bacteria-specific peptides and database search—for determining the species of bacteria present in a mixture. We identified the proteotypic peptides that were associated with specific bacteria by searching protein databases for the LC-MS/MS data. The retention time windows for specific peptide markers were used as an extra constraint so that the peptide markers of many bacterial species could be analyzed in a single LC-selective proteotypic-peptide analysis (SPA). We performed LC-MS/MS analyses on the proteolytic digest of cell extracts and monitored only the selected marker peptide ions at given elution time windows. The corresponding bacterial species could be characterized when the selected peptides that eluted at expected elution windows were identified correctly from the database. We managed to identify up to eight bacterial species simultaneously during a single LC-MS/MS analysis, as well as bacteria mixed in various abundances. Two marker ions having similar values of m/z, but obtained from two different bacterial samples, which would otherwise be selected as precursors within mass tolerance and would complicate the MS/MS data, were time-resolved using LC and then used to correctly identify their bacterial sources. The coupling of selective MS/MS monitoring with separation methods, such as LC, provides a highly selective and accurate analytical method for characterizing complex mixtures of bacterial species. Copyright © 2006 John Wiley & Sons, Ltd.