Measuring glucocorticoid metabolites in faeces has proven a useful, non-invasive method to monitor adrenocortical activity in several farm and wild species. Unlike plasma cortisol, whose sampling requires restraint and blood draws, faecal cortisol metabolites (FCM) may be particularly suitable for farmed silver foxes as these animals are sensitive to handling by humans. Prior to using FCM as a potential indicator of stress in silver foxes, however, a proper physiological and/or biological validation is required. Here, we determined FCM concentrations in 30 silver foxes (10 adult vixens, 10 juvenile females and 10 juvenile males) every alternate hour for 24 h after 1) an increase in cortisol induced by injection with synthetic ACTH (hereafter ACTH), and 2) a 2 min period of handling and restraint. Baseline FCM values, recorded every fourth hour for 24 h before the ACTH and handling treatments, served as controls. FCM values increased significantly following ACTH injection (P = 0.0001) and handling (P < 0.0001). The time to reach peak FCM concentrations after ACTH injection tended to differ between groups (P = 0.055) averaging (+/- SE) 11.0 +/- 1.04, 10.6 +/- 1.30 and 7.8 +/- 0.20 h for vixens, juvenile females and juvenile males, respectively. After handling, peak FCM values were reached after 10.1 +/- 0.55 h with no significant differences between groups. Peak concentrations averaged 2143 +/- 264 ng/g after the ACTH and 1008 +/- 128 ng/g after handling, compared to 475 +/- 48 ng/g for baseline levels. Peak FCM values tended to vary between individuals more in females than in males. Baseline FCM concentrations prior to handling were, unexpectedly, higher in more confident foxes (P = 0.004), a finding perhaps indicating a potential preparative role of cortisol in silver foxes. There was also a negative trend between foxes' confidence and their times to reach peak FCM concentrations after handling (P = 0.062), suggestive of a prolonged adrenocortical activation in more fearful individuals. Based on the rates that foxes produce faecal samples and the times to reach maximum FCM concentrations, we suggest a four hour delay to first faeces collection, before collecting samples every third hour the next 12 following hours to monitor elevations after an acute stressor. Our study confirms faecal cortisol metabolites as a valid indicator of adrenocortical activity in farmed silver foxes.