The development of non-invasive techniques to analyse physiological stress in mammalian species has revolutionised field-based endocrinology. However, careful validation of the methods used to determine faecal glucocorticoid metabolite (fGCM) and other hormone concentrations are required on a species- and sex-specific basis. In this study, we performed an adrenocorticotropic hormone (ACTH) stimulation test on four (two male and two female) captive vervet monkeys (Chlorocebus pygerythrus) to determine the most appropriate enzyme immunoassay (EIA) from a suite of available EIAs. Furthermore, we took advantage of a potentially stressful event in our wild vervet population from Samara Private Game Reserve, South Africa, to examine if an alpha–beta female rank reversal increases the physiological stress of those individuals directly involved, as well as other group members. Both our physiological and biological validation studies revealed that a cortisol assay was the most appropriate EIA for monitoring fGCM alterations in vervet monkeys. In addition, we found that the observed rank-reversal had no significant effect on the physiological stress levels of uninvolved group members. Our study highlights that physiological validation is imperative and, where possible, should be conducted in parallel with a carefully considered biologically-relevant test under natural conditions. Overall, our results provide a necessary step for future studies to examine physiological stress of vervet monkeys via fGCM monitoring by validating a suitable EIA for this species. This paves the way for future research into the health and welfare of both captive and wild vervet monkeys, and will allow researchers to assess the behavioural, social and ecological correlates of physiological stress levels of this species.