Abstract: Using indigo paste as raw material, indigo acidification substances, glucose reducing substances, and ethyl acetate extractives were prepared using three processes of hydrochloric acid acidification, glucose reduction, and ethyl acetate extraction, respectively. High performance liquid chromatography(HPLC), ultraviolet visible spectrophotometry(UV), and dual wavelength ultraviolet spectrophotometry(dW-UV) were established to analyze the contents of indigo and indirubin in samples from three processes. The optimal analysis methods for each of three samples were determined through Kolmogorov Smirnov test, comparison of Bland Altman deviation plots, and Spearman test. The results showed that the established HPLC, UV, and dW-UV methods were simple, accurate, highly sensitive, stable, and reproducible. Three methods for detecting the content of indigo extract components all followed the normal distribution, and there were significant differences between each the two measured values. The UV method was more suitable for analyzing the content of indigo in indigo acidified substances and indigo reducing substances. HPLC method was more suitable for measuring the content of indigo and indirubin in ethyl acetate extract.