ACE plays a significant physiological role in regulating blood pressure in the renin-angiotensin system. The antihypertensive potential of 3’,4’, Dihydroxy-3-5-dimethoxy flavone-7-O-β-rhamnose previously isolated from Peperomia pellucida (L.) Kunth. (Piperaceae) was assessed by inhibition of ACE inhibitory and in silico molecular docking. The ACE activity was evaluated by determining of hipuric acid level from the hydrolysis rate of the substrate N-hippuryl-L-histidyl-L-leucine  (HHL) and measuring its absorbance using a spectrophotometer. The structure of the pure compound, isolated from an aerial part of Peperomia pellucida has been established by spectral data (UV, IR, LC-MS, ¹H-NMR, ¹³C-NMR, and 2D-NMR) and compared with that reference. The experimental results were also confirmed by docking analysis to explore the binding conformations of isolated compound into the binding sites of ACE. The isolated compound possessed acceptable ACE inhibitory activity (IC₅₀ value of 7.72 μg/mL and binding energy of –6.92) in comparison with captopril (IC₅₀ value of 3.59 μg/mL and binding energy of –6.91). The results derived from in vitro and computational experiments, confirm the potential of the P. pelucida herb is good to use in traditional medicine to treat antihypertensive.