A reversed-phase high-performance liquid chromatography method for the quantitative analysis of valienamine in the microbial degradation of validamycin A, using a procedure for pre-column derivatization of valienamine with p-nitrofluorobenzene is described. Valienamine in the broth was first isolated with the ion-exchange method. The optimized conditions for the derivatization were the reaction time 30 min and reaction temperature 100℃. With the mobile phases consisting of acetonitrile-water (12∶88)(eluent A) and methanol (eluent B), the gradient was carried out with 100% of A for 15 min and then 100% of B for another 10min. The parameters in the process were the flow rate of the mobile phase 1.0ml/min, the injection volume 20μl, the column temperature 40℃ and wavelength of ultraviolet detection 398nm in all runs. A good linearity was found in the range of 0.5-150.0μg/ml. Both intra-and inter-day precisions of valienamine, expressed as the relative standard deviation, were less than 9.4%. Accuracy, expressed as the relative error, range from −0.5 to 2.7%. The mean absolute recovery of valienamine at three different concentrations was 94.2%. The method was proved suitable for the study on the process of microbial degradation of validamycin A to produce valienamine.