Abstract: A high-performance liquid chromatography(HPLC) method was established for determining contents of vitexin and isovitexin in the leaves, stems and roots of pigeonpea (Cajanus cajan(L.)Millsp). The analytes were separated on a HIQ Sil C18V(250 mm×4.6 mm,5 μm) column, using methanol-water-formic acid(35:64.74:0.26, volume ratio), as mobile phase, detection wavelength 330 nm, flow rate 1 mL/min, injection volume 10 μL, and column temperature 30℃. The calibration curves of vitexin and isovitexin showed good linearity within the ranges of 2-200 mg/L. For vitexin, relative standard deviation(RSD) of precision was not higher than 2.93%, RSD of repeatability was 2.91%, and recovery was 97.38%; for isovitexin, RSD of precision was not higher than 2.69%, RSD of repeatability was 3.37%, and recovery was 98.63%. Mass factions of vitexin and isovitexin in pigeonpea leaves, stems and roots were 0.768, 0.066, 0.183 mg/g and 0.799, 0.139, 0.013 mg/g, respectively. Pigeonpea leaves contain much higher amounts of vitexin and isovitexin than stems and roots, which could be developed as a good resource for obtaining vitexin and isovitexin on large scale.