Venoms have evolved at least 100 times in the animal kingdom, with their main biological roles being prey aquisition and self-defence. Snake venoms show extreme variability in composition at all taxonomic levels, both within and between species. The selective pressures underlying this variation have long been debated. Since snake venoms are used for both foraging and defence, we hypothesise that venom composition may represent a trade-off between lethal/immobilising predatory toxins and pain-inducing defensive toxins. Here, we use the Eurasian viper genus Vipera to test the hypothesis that snakes feeding on weakly defended prey, such as insects, should have venoms more adapted for defence than snakes feeding on well-defended prey such as rodents. We tested the nociceptor-activating (pain-inducing) capacity of the venoms of populations of the largely insect-eating Vipera ursinii complex and of the primarily mammal-eating V. ammodytes, V. aspis and V. berus. We used high-throughput Ca²⁺ imaging and deconvolution microscopy on F11 cells and native dorsal root ganglia cells, respectively, to test the algesic activity of the venoms of European vipers. Contrary to our expectations, none of the venoms resulted in nociceptor activation, irrespective of diet. We conclude that selection for defence is unlikely to have played a significant role in driving the evolution of venom composition in this genus.