Introduction: Hyphaene thebaica L. fruit is known in Egypt for its antihypertensive activity. However a standardized herbal extract/fraction was never prepared. Methods: A biologically guided fractionation was carried out in-vitro for the 50% and 70% ethanol extracts of Hyphaene thebaica L. fruit using the angiotensin converting enzyme (ACE) inhibition and renin inhibition assays. A validated reversed phase HPLC method was developed for the standardization of the active fractions. Results: The ethyl acetate fraction of the 70% extract contained higher percentages of the three bioactive markers chlorogenic acid, quercetin and apigenin (1.940 ± 0.140%, 2.994 ± 0.349% and 0.612 ± 0.0354%, respectively) relative to the ethyl acetate fraction of the 50% extract (1.384 ± 0.157%, 0.342 ± 0.0834% and 0.070 ± 0.00225%, respectively). The butanol fraction of the 70% extract was found to possess the highest antihypertensive activity (93.69 ± 5.695 % renin inhibition activity at 0.5 mg/mL and IC50 of 0.001436 +0.00044 mg/mL for ACE inhibition activity). A standard calibration curve for the three compounds was established at a concentration range of 0.1-50 μg/Ml, they showed good linearity with a correlation coefficient (R2) of (1.00, 1.00 and 0.999; respectively). A high degree of precision (relative standard deviation values <5%) was achieved. The limits of detection for the three compounds were 0.428, 0.368 and 0.849; respectively, while the limits of quantitation were 1.29, 1.11 and 2.57, respectively. Conclusions: Current results showed that the butanol fraction of the 70% extract revealed the highest antihypertensive activity through ACE and renin inhibition mechanisms. In addition, recorded observations concerning linearity of the used bioactive markers offer a support for the possible utility of the tested fractions as potent standardized antihypertensive drugs.