Knowledge of animals’ hormonal status is important for conservation studies in wild or semi-free-ranging conditions as well as for behavioural and clinical experiments conducted in laboratory research, mostly performed on rats and mice. Faecal sampling is a useful non-invasive method to obtain steroid hormone assessments. Nevertheless, in laboratory studies, unlike other contexts, faecal sampling is less utilised. One of the issues raised is the necessity to collect samples belonging to different animals, separately. Usually, researchers using faecal sampling solve this problem through the isolation of animals or taking the cage rather than single animal as unit of study. These solutions though, could lead to unreliable measurements, and cannot be applied in many studies. Our aim was to show the biological reliability of individual faecal corticosterone metabolite (FCM) assessments in socially housed male and female Wistar rats. We analytically validated the enzyme immunoassay kit used for FCM assessments. Then, we exposed the animals to two different stress stimuli that are known to activate the hypothalamus–pituitary–adrenal axis and the following release of corticosterone to biologically validate the EIA kit: environmental enrichment and predator odour. Individual faecal sampling from social animals was collected through short-time handling. The results demonstrated that both the stimuli increased FCM levels in male and female rats showing the reliability of EIA kit assessment and the applicability of our sampling method. We also found a diurnal rhythm in FCM levels. These results could help to increase the use of faecal hormone metabolite determinations in studies conducted on rats.