Identifying potential stressing factors and improving husbandry conditions of domestic animals is a major welfare issue. Plasma glucocorticoid concentrations are widely used to assess stress in an animal but the blood sampling procedure itself is not feedback-free. Quantification of stress hormones in the egg has been suggested as a non-invasive method to measure stress in laying hens and thus evaluate animal welfare. However, when using a new method, one important aspect is to prove its biological relevance and thus its ability to detect increased levels of stress hormones in the analysed material. As a sound biological validation for egg yolk is still lacking, we analysed chicken egg yolk for the presence of corticosterone. We performed HPLC separations and determined concentrations of immunoreactive corticosterone via enzyme immunoassay (EIA). In eggs of untreated chickens, we did not detect any immunoreactive corticosterone at the elution position of corticosterone, but found that the corticosterone antibody cross-reacts with progesterone and several other gestagens, which are present in the yolk in abundant concentrations. When eggs from laying hens with corticosterone implants and thus elevated plasma corticosterone concentrations were analysed, we found immunoreactive corticosterone as well as high amounts of cross-reacting gestagens, hampering quantification of corticosterone in yolk without previous clean-up. This study demonstrates that the high concentrations of gestagens present in the yolk cause a signal in a corticosterone immunoassay. Our findings therefore strongly suggest that measured concentrations of corticosterone in homogenised yolk may actually reflect to a large extend those of gestagens. They also illustrate possible confounding factors when measuring a certain steroid hormone out of a mixture of various unknown and potentially cross-reacting steroids. The study demonstrates the necessity of evaluating the biological relevance of a method whenever animal welfare is assessed via physiological parameters.