One of the target roads of the LIFE LINES Project is the Municipal Road 535 from the Montemor-o-Novo county. To establish the reference situation, a few field trips were conducted by the University of Évora during the months of November and December, where all the existing tunnels were identified, the road margins with suitable habitat for small mammals and the amphibian’s mortality hotspots. Also, with the help of Geographic Information System software all the water bodies in the vicinity of the road were identified. As a final result a total of 46 tunnels were indentified, 82 water bodies and 27 patches of good habitat for small mammals in the margins of the road. For amphibians were conducted 11 nocturnal transects where a total of 675 animals were found on the road. With these results a mitigation proposal was created and is currently under review by the Town hall of Montemor-o-Novo.