Coxiella burnetii, the causative agent of Q fever, poses a significant public health concern worldwide. Diagnosis primarily relies on serological tests. Traditional antigen production methods, typically involving embryonated hen eggs, are labor-intensive, costly, and require biosafety level 3 facilities. In this study, we tested inactivated whole-cell antigens (SAP9 and NMII/AP9) from C. burnetii strains grown in axenic media, offering a safer and more efficient alternative to egg-based production. These antigens were validated using an in-house ELISA method against human patient sera, demonstrating high sensitivity and specificity comparable to ELISA and to the Gold Standard, IFA commercial kits. Notably SAP9 and NMII/AP9 antigens showed no cross-reactivity with intracellular pathogens that cause illness with similar symptoms. This approach represents significant advancement in diagnostic antigen production for Q fever, facilitating cost-effective epidemiological studies and enhancing laboratory safety.