Despite the abundance of rock hyrax Procavia capensis within South Africa’s urban areas, there is not much information available about the effect of anthropogenic activities on rock hyrax wellbeing. To determine the potential impact of anthropogenic disturbance on adrenocortical activity, we conducted an ACTH challenge to identify a suitable enzyme-immunoassay (EIA) for measuring faecal glucocorticoid metabolite (fGCM) concentrations in the rock hyrax. This study identified an 11β-hydroxyaetiocholanolone EIA as the most suitable assay in this regard. The fGCM levels measured, indicate the physiological stress response in different rock hyrax populations, living in an area with varying degrees of anthropogenic activity (low, medium, high) within the National Botanical Garden of Pretoria, South Africa. The species’ habituation to human numbers (weekly mean number of people) was examined by determining individual flight initiation distance (FID). Seasonally, there were overall higher fGCM concentrations in late spring compared to winter. The fGCM concentrations, although not significantly different but possibly biologically relevant, in the section with the lowest anthropogenic disturbance were ~10% higher compared to those in the section with medium disturbance, and ~20% higher compared to those in the section with the highest disturbance. Animal FID did not differ significantly between seasons but they did differ significantly between sections, and decreased in accordance with fGCM concentrations. The non-invasive approach established in this study provides a foundation for assessing rock hyrax wellbeing, and can help better understand how anthropogenic presence is perceived as a stressor in this species.https://onlinelibrary.wiley.com/journal/1903220xam2023Mammal Research InstituteZoology and Entomolog