There has been conflicting evidence about the impact of visitors on zoo animals. The present study was designed to increase understanding of visitor effect by assessing the relationship between visitor numbers and activity in the hypothalamic–pituitary–adrenal (HPA) axis. An enzyme-immunoassay was successfully developed and validated to quantify urinary cortisol in spider monkeys. The physiological impact of visitors on the monkeys was then investigated by assessing the levels of urinary cortisol in samples collected when no visitors were in the zoo and throughout the year when visitor density fluctuated widely. Our results suggest that an increase in the number of visitors was associated with an increase in cortisol. This study supports previous behavioural research that visitors have a meaningful impact on primates in zoos. It also increases our understanding of visitor effect by assessing the relationship between absolute visitor numbers and an aspect of the animals’ physiology.