Yersinia pestis is the causative agent of bubonic and pneumonic plague, an acute and often fatal disease in humans. In addition to the risk of natural exposure to plague, there is also the threat of a bioterrorist act, leading to the deliberate spread of the bacteria in the environment or food. We report here an immuno-liquid chromatography-tandem mass spectrometry (immuno-LC-MS/MS) method for the direct (i.e., without prior culture), sensitive, and specific detection of Y. pestis in such complex samples. In the first step, a bottom-up proteomics approach highlighted three relevant protein markers encoded by the Y. pestis-specific plasmids pFra (murine toxin) and pPla (plasminogen activator and pesticin). Suitable proteotypic peptides were thoroughly selected to monitor the three protein markers by targeted MS using the selected reaction monitoring (SRM) mode. Immunocapture conditions were optimized for the isolation and concentration of intact bacterial cells from complex samples. The immuno-LC-SRM assay has a limit of detection of 2 × 10(4) CFU/mL in milk or tap water, which compares well with those of state-of-the-art immunoassays. Moreover, we report the first direct detection of Y. pestis in soil, which could be extremely useful in confirming Y. pestis persistence in the ground.