The laboratory diagnosis of rickettsioses is based on serology (reference method), cell culture and/or molecular tools. However, the main drawback of serology is its incapacity to provide identification of Rickettsiae at the level of species. The aim of this study was to propose the versatile protein markers able to discriminate the patients with murine typhus from those with Mediterranean spotted fever. We have cloned and expressed 20 proteins of Rickettsia prowazekii and Rickettsia rickettsii, respectively, using the GATEWAY approach. These recombinant proteins were screened by ELISA with sera of infected patients with Rickettsia typhi and Rickettsia conorii, respectively. We identified several potential markers which allowed infection due to R.typhi to be discriminated from those due to R.conorii. However, the values of test-operating parameters were not sufficient for its 'routine' clinical use. Our diagnostic test requires further optimization for be applied as a point-of-care strategy in the management of patients with suspected cases of rickettsiosis.