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The handling of highly pathogenic viruses, whether for diagnostic or research purposes, often requires an inactivation step. This article reviews available inactivation techniques published in peer-reviewed journals and their benefits and limitations in relation to the intended application. The bulk of highly pathogenic viruses are represented by enveloped RNA viruses belonging to the <i>Togaviridae</i>, <i>Flaviviridae</i>, <i>Filoviridae</i>, <i>Arenaviridae</i>, <i>Hantaviridae</i>, <i>Peribunyaviridae</i>, <i>Phenuiviridae</i>, <i>Nairoviridae</i> and <i>Orthomyxoviridae</i> families. Here, we summarize inactivation methods for these virus families that allow for subsequent molecular and serological analysis or vaccine development. The techniques identified here include: treatment with guanidium-based chaotropic salts, heat inactivation, photoactive compounds such as psoralens or 1.5-iodonaphtyl azide, detergents, fixing with aldehydes, UV-radiation, gamma irradiation, aromatic disulfides, beta-propiolacton and hydrogen peroxide. The combination of simple techniques such as heat or UV-radiation and detergents such as Tween-20, Triton X-100 or Sodium dodecyl sulfate are often sufficient for virus inactivation, but the efficiency may be affected by influencing factors including quantity of infectious particles, matrix constitution, pH, salt- and protein content. Residual infectivity of the inactivated virus could have disastrous consequences for both laboratory/healthcare personnel and patients. Therefore, the development of inactivation protocols requires careful considerations which we review here.