Heat stress (HS) poses a critical concern for the global poultry industry. To better understand its impact and mechanism on poultry species, as well as to develop effective HS mitigation options, it is crucial to validate reliable and effective biomarkers of HS. In this study, broiler chickens (96 mixed-sex, 1-day-old) were randomly assigned to 2 environmental conditions: HS at 31°C for 8 h/d versus a thermoneutral condition (TN) at 24°C ± 0.2 throughout the day, from d 21 to d 28. After 7 d of cyclic HS, blood serum samples and the urate sphere (the white portion of the chicken excreta) were collected to measure specific HS biomarkers, including corticosterone, heat shock protein 70 (HSP70), Triiodothyronine (T3), and urate sphere corticosterone metabolite. The results showed that the concentrations of HSP70, T3, and serum corticosterone were not significantly different (P > 0.05) between the heat-stressed and thermoneutral birds in this study. However, heat-stressed birds recorded higher (P < 0.01) concentration (pg/mL) of urate corticosterone metabolite compared to TN birds. Data presented here thus provide preliminary evidence suggesting the use of non-invasive urate sphere corticosterone metabolite as a heat stress biomarker in poultry.