A chemical fingerprint method for methanolic extract of seeds of Psoralea corylifolia Linn. was developed for Quality Control analysis by Ultra Performance Liquid Chromatography with Photodiode Array Detector (UPLC-PAD). Psoralea corylifolia Linn., seeds collected from Pharmacy, Central Research Institute of Unani Medicine, Hyderabad and extract was analyzed by using a reverse-phase Waters Acquity BEH C 18 column (50 mm × 2.1 mm, 1.7 μm). Among mobile phases investigated such as methanol-water, acetonitrile-water, and acetonitrile-buffer (containing Orthophosphoric acid), the water (A) - acetonitrile (B) system was the ultimate choice. Gradient elution was essential with gradient mobile phase consisted of 0.1%OPA in (water: Acetonitrile : 10:1) (A) and acetonitrile (B) using a gradient program at a flow rate of 0.5 mL/min with detection at a wavelength of 254 nm. The chromatographic fingerprints showed different chemical constituents qualitatively in Psoralea corylifolia Linn. seeds, out of which psoralen peak identified corresponding to the retention time of standard Psoralen and further confirmed by UV spectrum. The Psoralen content in seeds extract of Psoralea corylifolia Linn. which is an active principle is accurately determine corresponding to that of standard Psoralen with shorter analysis time.