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Understanding the pathogenesis of infectious diseases requires comprehensive knowledge of the proteins expressed by the pathogen during in vivo growth in the host. Proteomics provides the tools for such analyses but the protocols required to purify sufficient quantities of the pathogen from the host organism are currently lacking. Here, we present a rapid immunomagnetic protocol for the separation of Francisella tularensis, a highly virulent bacterium and potential biowarfare agent, from the spleens of infected mice. In less than one hour, bacteria can be isolated in quantities sufficient to carry out meaningful proteomic comparisons with in vitro grown bacteria. Furthermore, the isolates are virtually free from contaminating host proteins. Two-dimensional gel analysis revealed a host induced proteome in which 78 proteins were differentially expressed in comparison to in vitro grown controls. The results obtained clearly demonstrate the complexity of the adaptive response of F. tularensis to the host environment, and the difficulty of mimicking such behavior in vitro.