AbstractFLASH radiotherapy is a novel technique that has been shown in numerous preclinicalin vivostudies to have the potential to be the next important improvement in cancer treatment. However, the biological mechanisms responsible for the selective FLASH sparing effect of normal tissues are not yet known. An optimal translation of FLASH radiotherapy into the clinic would require a good understanding of the specific beam parameters that induces a FLASH effect, environmental conditions affecting the response, and the radiobiological mechanisms involved. Even though the FLASH effect has generally been considered as anin vivoeffect, studies finding these answers would be difficult and ethically challenging to carry out solely in animals. Hence, suitablein vitrostudies aimed towards finding these answers are needed. In this review, we describe and summarise severalin vitroassays that have been used or could be used to finally elucidate the mechanisms behind the FLASH effect.