Acute tryptophan depletion (ATD) is a valuable non-invasive nutritional tool in human and rodent research to study dysfunctions of the serotonergic system and related behavioral disorders. Serotonergic dysfunction is thought to be involved in the pathology of feather pecking behavior of laying hens, one of the most relevant welfare and production issues in modern intensive egg-production systems. ATD temporarily compromises the influx of tryptophan (TRP) across the blood brain barrier which reduces central availability of TRP, the substrate for serotonin (5-HT) synthesis. However, ATD has never before been developed and evaluated in birds. We hereby report that ATD in laying hens effectively depletes plasma levels of TRP to 50% of the baseline concentration, 4 hours after administration. Furthermore, ATD reduces the ratios of TRP towards aromatic amino acids (AAA) by 60% and the ratio of TRP towards large neutral amino acids (LNAA) by 70%, three hours after administration. Further studies will be needed to determine the effects of peripheral depletion on brain TRP and 5-HT levels in birds. However, our study showed for the first time in an avian species that ATD causes lowering of plasma TRP and the ratio in plasma of TRP towards other AAA or LNAA.