In the recent past, tissue materials such as hair/coat and nails/claws have proved to be useful for the study of long-term hormonal changes in humans and animals and shown to be advantageous in terms of being collectable without invasiveness, with a benefit in terms of animal welfare. However, studies using these tissue materials in cats are scarce, especially on sexual hormone measurement. In this study, the concentrations of 17-β-estradiol (E2) and testosterone (T) were assessed in 20 male and 18 female domestic postpubertal cats at the time of neutering/spaying during the breeding season. Hormones were measured in coat shaved from the forearm (ACOAT) and from the surgical area (SCOAT); claws were collected from the forearms (CLAWS) and the dewclaws (DCLAWS). Although all these tissue materials were shown to be useful for E2 and T long-term measurement, only T concentrations were higher (p < 0.001) in males from both ACOAT and SCOAT samples when compared to females and therefore useful for distinguishing between the two sexes. Within each sex, E2 and T concentrations can be assessed on coat, but also on the dewclaws, providing an alternative, practical, matrix for sexual steroid measurement in postpubertal cats during the breeding season.