A method for biomarker candidate discovery and strain level pathogen characterization using liquid chromatography/mass spectrometry (LC/MS) with electrospray ionization is described. This method was applied to two pathogenic Clostridium species: C. difficile and C. perfringens. Seven marker proteins per species (fourteen total) were successfully implemented to speciate unknowns during a blind study and could enhance serological and genetic approaches by serving as new targets for detection. Two sets of C. perfringens isolates that were 100% similar by pulsed-field gel electrophoresis (PFGE) were distinguished using LC/MS, demonstrating the high specificity of this approach. The use of LC/MS is less labor intensive than PFGE, affords greater specificity than real-time PCR, and requires no primers or antibodies.