Since the untargeted metabolomics analysis used self- controlled design, the proposed biomarkers were further validated by control samples from other origins using UHPLC–MS/MS detection. The treated samples of 5 and 13 withdrawal days were selected, and 20 negative urines from different local farms were collected. Fluorometholone acetate was up-regulated by 2.7 and 1.85-fold in 5 and 13 withdrawal days urine samples, respectively, while 2-indolecarboxylic acid was down-regulated by 2.17 and 1.48-fold. These results were consistent with untargeted analysis.