Abstract <p>Noninvasive methods for assessing animal hormonal status areincreasingly applied in ecophysiological studies, which is particularlyimportant for rare and vulnerable species. In vertebrates, the hypothalamic–pituitary–adrenal(HPA) axis and its effector hormones, glucocorticoids, play a central rolein regulating physiology and behavior in response to environmentalchallenges, including various stressors. This study aimed to validatea noninvasive method for assessing HPA axis activity by measuringcorticosterone metabolites in the droppings of two migratory passerinebird species, the European robin (<i>Erithacusrubecula</i>) and the garden warbler (<i>Sylviaborin</i>), using EIA-4164 kits (DRG International Inc.,USA). Physiological validation using an ACTH challenge induced asignificant increase in plasma corticosterone concentrations, followedby an increase in corticosterone metabolite levels in droppings.In both species, metabolite levels peaked within three hours afterACTH injection, consistent with gut passage time. Experimental procedures(blood sampling and ACTH injection) temporarily suppressed feedingactivity in some birds, leading to a negative correlation betweendroppings mass and corticosterone metabolite concentrations, i.e.,metabolite concentrations were higher in samples with lower droppingsmass. Given the considerable variation in feeding intensity among experimentalbirds, expressing results as the absolute amounts of corticosteronemetabolites excreted over fixed time intervals (excretion rate)reduced bias associated with variation in droppings mass and provideda more reliable assessment of hormonal status than concentration-basedmeasures. This study validates the EIA-4164 kit for noninvasivemonitoring of HPA axis activity in two small migratory passerine speciesand highlights the importance of accounting for the mass of droppingsto improve the accuracy of hormonal status assessments.</p>