A reliable and complete inactivation is an indispensable premise for any concentration of rickettsiae or for the development of diagnostic strategies based on their antigens. This study deals with the testing of methods to inactivate rickettsiae. Rickettsia honei was used as a model organism. The inactivating potency of formalin, Qiagen® antiviral lysozyme (AVL) buffer, heating to 56 °C, and β-propiolactone was analyzed in cell culture. The inactivation limits for rickettsiae were 0.1% formalin about 10 min, Qiagen AVL buffer about 5 min, 56 °C about 5 min, 0.125% β-propiolactone about 1 h, and 0.0125% β-propiolactone overnight. The interpretation was limited by cytotoxic effects of the inactivation procedures and by the culturally achievable rickettsial density in the cell culture supernatants that were used for the inactivation experiments. Reliable modes of inactivation were identified, allowing for the secure handling of rickettsial antigens for diagnostic purposes.