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A cortisol metabolite, 11-ketoetiocholanolone (11-k), is widely used in monitoring stress in several vertebrates, and can be detected by immunoassay. However, these assays have certain limitations with respect to specificity. Also, differences in the excretion of faecal glucocorticoid metabolites (FGM) among species and even between sexes make validation necessary in each case. Therefore, our aims were, first, to develop and validate a high-pressure liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) methodology for monitoring 11-k in faeces of Iberian red deer (Cervus elaphus hispanicus), and second, to investigate the capability of our method to determine variations of this FGM in a longitudinal study. Third, and finally, we assessed the correspondence between faecal 11-k concentrations and plasma cortisol. An adrenocorticotropic hormone (ACTH) test was performed on six red deer stags translocated and kept in captivity for a week and faecal samples were collected twice a day. One single blood and faecal sample from another seven stags was also collected after 2 weeks in captivity. The results of the longitudinal study showed a first peak in 11-k 36 h after the ACTH test and handling, and a second peak at 120 h of being kept indoors. Maximum concentrations of 11-k ranged from 22.71 to 375.68 ng/g. In the second stag group, 11-k concentrations of 25.09 ± 20.53 ng/g had a correlation of r2 = 0.88 with the concentration of plasma cortisol, which was 54.6 ± 55.1 ng/mL. This technique is capable of detecting changes in the concentrations of faecal 11-k. The values determined have a good correlation with the cortisol concentration in blood, and we also detected differences in different individuals’ responses to the same stressors.

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