Personnel deployed to remote areas during infectious disease outbreaks have limited access to mechanical and chemical inactivation resources. The inactivation of infectious agents present in diagnostic samples is critical to ensure the safety of personnel and the containment of the disease. We evaluated the efficacy of thermal inactivation (exposure to 56°C for 1 hour) and chemical inactivation with 0.5% Tween-20 against a high titer of Ebola virus (species Zaire ebolavirus) variant Makona in spiked human serum samples. No surviving virus was revealed by a 50% tissue culture infective dose assay after the combined treatment under laboratory conditions. In-field use of this inactivation protocol during the 2013–2016 West Africa Ebola outbreaks demonstrated readily detectable levels of immunoglobulin G and/or immunoglobulin M in human plasma samples after treatment.