Background and Purpose: Boerhavia diffusa is an herbaceous member of the Nyctaginaceae family which has been widely used in folk medicine to treat several illnesses. Experimental Approach: A validated HPTLC method has been newly developed for the quantification of eupalitin 3-O- β-D-galactopyranoside in hydroalcoholic extracts of B. diffusa. A three-level factor Box-Behnken statical design was used for optimization, extraction time (min), temperature (ºC), and methanol: water ratio (% v/v) are independent variables while bioactive compounds as the dependent variable. This study was aimed to investigate the pretreatment of HepG2 cells with hepatoprotective agents against the damage induced by galactosamine (GalN) and evaluate in vivo antihypertensive activity of eupalitin 3-O- β-D-galactopyranoside in rats by prednisolone inducing hypertension. Key Results: The separation was achieved on silica gel 60F254 HPTLC plate using: toluene: acetone: water (5: 15:1) as mobile phase. Densitometric analysis of eupalitin 3-O- β-D-galactopyranoside was carried out in the absorbance mode at 366 nm. A study has shown that optimized eupalitin 3-O-β-D-galactopyranoside can be used as an antihypertensive drug in rats by prednisolone inducing hypertension and In-vitro Hepatoprotective Activity of optimized eupalitin-O- β-D-galactopyranoside was checked in HPG2 cell line compared with silymarin by ANOVA technique. Conclusion and Implications: It is concluded that A new method for a method of validation and quantification of eupalitin-O-β-D-galactopyranoside in Boerhavia diffusa has been developed, In vivo antihypertensive and in-vitro hepatoprotective activity of optimized eupalitin O- β-D-galactopyranoside was done in intoxicated rats and in HepG2 cells induced by galactosamine (GalN).