Facilitating successful movements is fundamental to sustaining wildlife populations. Roads impede successful movements via barrier effects and as direct mortality sources. Quantifying such impacts is challenging but desirable for mitigation efforts and justifying associated costs. We have initiated a before-after-control-impact study design to quantify the impacts of a 4-lane highway in southern New York on local wildlife distributions, movement behaviors, gene flow, and mortality. Four years of camera trap surveys across our study area suggest distribution impacts from the highway, with some species detected only on one side of the highway (e.g., fisher [Pekania pennanti] and porcupine [Erethrizon dorsatum]). GPS-collar tracking of bobcats [Lynx rufus] reveals modified behaviors adjacent to major roads as well as preferred locations for road and highway crossing, both under and over. Observed highway crossing behaviors spurred monitoring efforts of eight culverts beneath the highway via a 4-camera trap per culvert design, suggesting species-level patterns in willingness to approach the highway, frequency of ‘turn back’ behavior, and likelihood of successfully crossing, including associations with culvert attributes and human use frequency. Deer-vehicle collision reports suggest >2.7 deer-vehicle collisions per mile annually (since 1998), however internal records from the NYS Thruway suggest the number of white-tailed deer [Odocoileus virginianus] carcasses removed is 30% greater (i.e., 3.5 deer per mile during 2021). We have initiated our own semi-weekly road mortality survey to bolster these data finding an estimated 39.1 roadkills per mile annually. Fecal DNA sampling of four carnivore species (i.e., black bear [Ursus americanus], coyote [Canis latrans], red fox [Vulpes vulpes], and bobcat) is underway to quantify landscape-level gene flow. Plans for systematic surveys for small mammals, bats, and terrestrial herpetofauna are being developed for initiation in summer of 2023. Our goal is to robustly quantify and justify the need for improved connectivity across this major highway, ultimately guiding the design and implementation of rigorous mitigation plans to restore functional connectivity to this biologically rich but fragmented landscape.