Abstract: A method of screening for in vitro metabolites of meisoindigo using RP-HPLC-DAD (photodiode array detector) with gradient elution was reported. Meisoindigo, a new structural antitumor agent, was metabolized by rat liver microsomes in vitro. The extract of the metabolic sample was dissolved in MeOH-acetone (9: 1). An aliquot of the solution was injected onto a 5 μm YWGC18 (ODS) column (15 cm×4.0 mm I D)and separated with H₂O (0.05% H₃PO₄)—MeOH in linear gradient elution. The flow rate was 0.5 ml/min and the oven temperature was 40℃. Two simultaneous monitoring wavelengths (270 and 380 nm) were used in order to avoid detective losses of unknown metabolites. The spectral recording range of chromatographic peaks was set from 220 to 440 nm. After comparison of the chromatograms and the spectra of chromatographic peaks, ten metabolites were screened out which gave similar spectrum. Of them, the main chromatographic peaks (No. 7, 8) were confirmed as two monohydroxy metabolites by EI-MS. Their relative contents were calculated by area normalization method. The experimental results show that this method is characteristic of highly effective separation and specific detection. The whole analytical course can be completed in less than two hours. The effects of different elution models, detective parameters and components of mobile phase on the analytical results were discussed.