At least 10 percent of the approximately 300 species of the order Falconiformes are listed as being globally threatened. The present work describes the seminal characteristics of three diurnal and one nocturnal raptor species. Semen was collected from clinically healthy Accipiter nisus (n = 1), Falco subbuteo (n = 6), and Falco tinnunculus (n = 5) adult males that were housed at the ‘Centro Animali Non Convenzionali’ of the Department of Veterinary Sciences of the University of Turin. The semen was collected after a period of recovery and before their release as well as from seven Bubo bubo males bred in captivity as part of a raptor conservation project. All the potential semen donors were trained in semen collection during the breeding season via a ritualized procedure. Ejaculation was achieved using a massaging technique. Each sample was evaluated for volume, degree of contamination, and spermatozoa concentration. The semen motility and kinetic parameters were assessed on diluted semen (modified tyroides albumin lactate pyruvate, pH 7.5, temperature 37.5 °C) using a computer-aided sperm analyzer. Semen collection was successful in all the diurnal species and in five B bubo individuals. The sperm motility and sperm kinetic parameters were very variable both among and within species. In contrast with previous studies that involved raptors bred in captivity and imprinted on humans, we worked with wild birds and attempted to overcome the problem of poor semen quality, which is strongly influenced by stress, by adopting a ritualized procedure that has never been reported for semen collection purposes.