Aim: The study aims screening of phytochemicals and standardization of extract of Pedalium murex Linn. fruits. Methods: The extraction was carried out by maceration technique using 70% ethanol and 30% water. The aqueous ethanolic extract obtained was estimated for total phenolic and flavonoid content against the standard gallic acid and quercetin. Liquid –liquid partitioning method using solvents n-hexane, chloroform, ethylacetate provided the different fractions of aqueous ethanolic extract. Standardization was done with highly sensitive, accurate, easy and cost effective high performance liquid chromatographic method. Results: The preliminary phytochemical screening of aqueous ethanolic extract of P. murex fruits revealed the presence of alkaloids, saponins, tannins, flavonoids, sugar, glycosides, phenols and sterols. The total phenolic content determined using linear regression equation (y = 0.0123x - 0.0096 R² = 0.9962) was found to be 27.1± 0.72 mg/g equivalent of gallic acid while the flavonoid content was expressed as 17.6 ± 0.79 mg/g equivalent of quercetin. The mother extract and the aqueous fraction revealed peaks complementary with the chromatogram of standard quercetin addressing presence of quercetin. Conclusion: The study helped in developing the phytochemical profile of the extract and the standardization performed in the study confirmed the presence of quercetin in the mother extract and fractions.