The non-invasive assessment of adrenocortical activity by measuring faecal glucocorticoid metabolites (FGMs) in faeces is currently a widely used method, especially in exotic animals. The aim of our study was to biologically validate the suitability of six different enzyme immunoassays (EIAs) to quantify changes in FGM concentrations after a known strong stress stimulus (capture, sedation, blood collection for serological examination and tuberculination) in ring-tailed lemurs (Lemur cotta). Faecal samples were collected from six animals (four males and two females) twice daily spanning from two days before until three days after the exposure to the stress stimulus. The faecal samples obtained were extracted and the supernatants were measured for immunoreactive FGM concentrations using six different EIAs, namely cortisol, corticosterone, 11-oxoaetiocholanolone 1, 11-oxoaetiocholanolone II, 11 beta-hydroxyaetiocholanolone and 5a-pregnane-3 beta,11 beta,21-triol-20-one EIA. The baseline levels, peak levels (absolute concentration and percentage increase), the duration of the peak and lag time of the peak were calculated and statistically evaluated. The FGM concentrations started to increase on average 23 hours after the exposure to the stress stimulus and returned to pre-stress levels on average within 36 hours. Our results suggest that the two group-specific 11-oxoaetiocholanolone EIAs and the 11 beta-hydroxyaetiocholanolone assay are generally suitable for the monitoring of stress response in ring-tailed lemurs.