Abstract Yin and Yang are the two counter‐balancing aspects in ancient Chinese philosophy. In traditional Chinese medicine, Yin deficiency syndrome (YDS) is a common sub‐health state with complex causes. While the syndrome may be treated to various degrees of effectiveness with traditional Chinese medicine, efficient modern methods are yet to be developed for diagnosing and treating the YDS. Here we performed a metabolomics study on YDS in rats. Serum metabolites in rats were analyzed using ultra‐performance liquid chromatography‐mass spectrometry (UPLC–MS) method to identify potential biomarkers for YDS. The rats were divided randomly into the healthy control group, the untreated YDS group, and the anemarrhena treated YDS group. Compared with the control group, significant increase in the metabolites such as dihydrotestosterone (DHT) and 5β‐DHT, 4‐imidazolone‐5‐propanoate, 4‐(L‐alanin‐3‐yl)‐2‐hydroxy‐cis,cis‐muconate 6‐semialdehyde, and 5‐(L‐alanin‐3‐yl)‐2‐hydroxy‐cis,cis‐muconate 6‐semialdehyde were observed in the serum of untreated YDS group, which returned to normal in the anemarrhena treated group. Therefore, these metabolites may serve as potential biomarkers for YDS, and may facilitate the diagnosis and treatment of YDS.