Stress contributes to significant morbidity and mortality in birds impacting growth, reproductive activity, healing, and immune function. Aquapuncture has been used anecdotally for the treatment of stress-associated clinical signs (Shen Disturbance) in birds. The goal of this study was to evaluate the effects of cyanocobalamin (vitamin B12) aquapuncture at An-shen on salivary corticosterone concentrations, stress-induced behaviors, and heart rate over a 60-minute period in domestic chickens (Gallus gallus domesticus) and Japanese quail (Coturnix japonica). A group of 21 chickens and 10 quail were randomly assigned to either a Treatment Group, receiving bilateral An-shen aquapuncture with a 1:1 vitamin B12 and 0.9% sterile saline solution, or a Control Group, receiving no treatment. Physical examinations were performed on each animal, which served as the initial stressor followed by aquapuncture in the Treatment Group. Saliva samples were obtained at 0-, 15-, 30-, 45-, and 60-minutes post-examination. Animals were restrained in hand for the duration of the 60-minute experiment to provide a consistent stressor. Samples were frozen and salivary corticosterone concentrations were quantified using commercially available enzyme immunoassay (EIA). Subjective sedation was observed in the majority of treated chickens (8/11; 72.7%) and quail (4/5; 80%). In all treated birds, there was a significantly larger decrease in heart rate when compared to control animals. While quail had limited saliva yield, precluding meaningful analysis, salivary corticosterone proved to be an effective minimally invasive biomarker in chickens, with median salivary concentrations consistently lower than those of controls at each time point. Collectively, these findings indicate that bilateral An-shen aquapuncture resulted in sedation, decreased heart rate, and lower salivary corticosterone concentrations consistent with decreased stress, supporting its potential as a practical and clinically relevant intervention for stress reduction in the studied species.