Non-invasive techniques for the monitoring of animal well-being, such as faecal hormone analysis, are increasingly becoming some of the most desirable methods for addressing practical conservation questions. Despite the widespread use of faecal hormone measurements for monitoring responses to stressors and its known applicability to African wild dogs Lycaon pictus, the potential influence of time of collection post-defaecation on stress-steroid concentrations in faecal matter has not yet been investigated. In the present study, we determined the rate at which African wild dog faecal glucocorticoid metabolite (fGCM) concentrations change over a 16-day period post-defaecation, in order to provide recommendations for best sampling practice. No significant changes in fGCM concentrations were found for the first 48 h post-defaecation. However, an approximately 30% increase in fGCM concentrations were already noted between day 1 and day 2, followed by a significant 150% increase at 96 h post-defaecation. We therefore suggest that respective faecal material should be collected within the first 24 h post-defaecation to ensure the reliability of fGCM analysis. In addition, we collected baseline data denoting the fGCM concentrations of captive African wild dogs sampled across three South African captive sites. Determined baseline fGCM concentrations differed between African wild dogs at the sites sampled. These data could be used in future studies aimed at identifying the key stressor complexes perceived by captive African wild dogs in order to improve management strategies.