Proteomics have been used widely to study proteins in complex materials such as cells, body fluids, tissues, and organisms. Application of advance proteomic techniques for the characterization of disease-specific proteins may provide information for the detection of potential infectious agents. In this report, two proteomics techniques, a two-dimensional differential gel electrophoresis (2D-DIGE) and a one-dimensional gel electrophoresis and one-dimensional liquid chromatography coupled with mass spectrometry (GeLC-MS/MS), were applied for investigating viral proteins from cultured cells inoculated with a clinical sample. The 2D-DIGE method identified five viral proteins of vaccinia virus that are only present in infected cells, these results are in agreement with findings determined by genome based methods. The GeLC-MS/MS method identified eight vaccinia virus proteins out of 428 proteins detected in the sample. These results demonstrate that proteomic techniques can be used effectively for the detection of infectious agents. Given that the methods are capable of applying to proteins without a prior knowledge of the pathogen present, proteomics has a potential of being developed as a molecular tool for pathogen discovery, and disease diagnosis of emerging infectious diseases and for bioterrorism defense.