Non-invasive methods to monitor adrenocortical activity need thorough validation. Besides analytical issues, the ability of the chosen test system to detect small changes in hormone concentrations triggered by stress perception must be evaluated. In this study, we biologically validated a previously established enzyme immunoassay (EIA) for corticosterone metabolites (CM) in chicken droppings. Adult laying hens were subjected to one hour of transport and in another experiment to 10 min of restraint. Droppings were collected subsequently after each stressor, over 36 h in total. Additionally, we analysed droppings that were collected after the birds first arrived at the experimental site and encountered unfamiliar housing conditions. Transporting the hens caused significantly increased mean CM concentrations for 3 h (328 nmol/kg droppings; p = 0.02) compared to baseline values (101 nmol/kg), whereas after 10 min of restraint, elevated levels of CM were not detected (166 nmol/kg; p = 0.87). When chickens were confronted with the new environment, CM concentrations stayed significantly elevated over the whole 36 h sampling period (> 313 nmol/kg; p < 0.05). In conclusion, this method is suitable to evaluate disturbances such as transport non-invasively in chickens.