Clostridium difficile infections are the leading cause of diarrhea associated to the use of antibiotics. During infection, C. difficile initiates a sporulation cycle leading to the persistence of C. difficile spores in the host and disease dissemination. Nowadays, the development of vaccine and passive immunization therapies against C. difficile have focused on toxins A and B. In the present study, we used an immunoproteome-based approach to identify immunogenic proteins located on the outer layers of C. difficile spores as potential candidates for the development of immunotherapy and/or diagnostic methods against this devastating infection. Experimental design. To identify potential immunogenic proteins on the surface of C. difficile R20291, spore coat/exosporium extracts were separated by two-dimensional electrophoresis (2-DE) and analyzed for reactivity against C. difficile spore-specific goat sera. Finally, the proteins present at the spot of the interest were in-gel digested with chymotrypsin, and the peptides generated were separated by nanoUPLC followed by MS/MS using a Quad-TOF MS, and corroborated by Ultimate 3000RS nano UHPLC coupled to QExactive Plus Orbitrap MS.