Reptile populations are suffering substantial global losses and roads are identified as one of the leading threats to their persistence. Currently, efforts to mitigate this threat are being implemented with various levels of success. I studied the effectiveness of exclusion structures (i.e., fencing) at preventing reptiles from gaining access to the road, and reducing road mortality. I also examined if population connectivity structures (i.e., ecopassages) were effective at reducing habitat and population fragmentation and allowing individuals to access habitats, resources, and mates on both sides of a major road (4 lane highway). I found that the fence was ineffective at preventing reptiles from gaining access to the road; however, reptiles were observed using the ecopassages to cross the road. Behavioural trials testing painted turtles’ (Chrysemys picta) willingness to use an ecopassage demonstrated that refusal was twice more likely than use of an ecopassage. I also examined the potential for roads to pose a physiological threat to roadside populations of reptiles by examining corticosterone (CORT), a stress hormone linked to negative health effects in cases of elevated levels over the long-term. To assess if individuals living near a major road had higher CORT levels than individuals from a less impacted population, I developed a novel means of measuring CORT from painted turtle claws in partnership with Dr. Gabriela Mastromonaco (Toronto Zoo). With long-term CORT levels considered as a proxy for chronic physiological stress, I did not find evidence that populations near roads had altered stress levels. However, this seminal study will provide the framework for further examination of more species, including species-at-risk, and a better understanding of effects of anthropogenic environments on wildlife health. As road ecologists strive to expand our understanding of the threats roads pose to reptiles, it is important that this field spans multiple disciplines, so that we can both understand the direct and indirect threats that roads cause and develop effective mitigation that preserves biodiversity within our anthropogenic landscape.