Abstract: Valienone is a significant natural carbasugar member of the C7-cyclitol family as a valuable precursor for glycosidase inhibitor drugs. It is an intermediate of validamycin A biosynthesis pathway and exhibits minimal accumulation in the fermentation broth of the natural Streptomyces producer. A quantitative analytical method is crucial for the development of a breakthrough microbial process overcoming the consumption of the natural metabolic flux. The present study was designed to develop a pre-column derivatization high-performance liquid chromatography method for quantification of valienone and to help establish a straightforward fermentation process for valienone production by metabolically engineered Streptomyces hygroscopicus 5008. Valienone was derivatized by 2, 4-dinitrophenylhy-drazine (DNPH) in 10 mmol·L^-1 H₃PO₄ at 37℃ for 45 min and the derivatives were separated on Eclipse XDB-C₁₈ (5 μm, 4.6 mm×150 mm) column at 30℃ eluted with 50% acetonitrile for 18 min. The derivatives were detected by diode array detector at 380 nm and the configurations of the derivatives were determined by compu-tational studies. The method was shown to be effective, sensitive, and reliable. Good linearity was found in the range of 5-2 000 μg·mL^-1. The intra-and inter-day precisions were 1.1%-2.7% and 1.7%-2.2%, respectively. The absolute recovery of the spiked samples was 97.2%-102.6%. To date, this is the first reversed-phase high-performance liquid chromatography detection method for valienone in microbial culture medium. This method successfully helped evaluate the valienone production capability of the engi-neered Streptomyces hygroscopicus 5008 and could be promising for C7-cyclitol profiling of different engineered mutants combined with the metabonomics methods.