Peperomia pellucida L. Kunth has been reported to have biological activities such as antihypertensive and antioxidants. Herbal materials susceptible to contamination during processing and storage which can shortens their shelf life. Gamma-irradiation is an non-thermal process that has been used as a method for preservation. This research aimed to analyze the effect of gamma-irradiation on inhibition activity of ACE, antioxidant activity, total phenol content, total flavonoid, and TLC profiles of ethanol 70 % extract P. pellucida L. Kunth herbs. Dried P.pellucida Kunth herbs were extracted by reflux method using ethanol 70%. These extracts were irradiated with ⁶⁰Co gamma rays applying doses of 2,5; 5; 7,5; dan 10 kGy. Total phenolic content was measured by Folin-Ciocalteu method, total flavonoids were tested using AlCl₃-colorimetry while ACE inhibitory activity was evaluated using ACE-kit-WST, and antioxidant activity was measured by DPPH radical reduction method. No significant differences were noted in the inhibition activity of ACE while total flavonoids showed a significant decrease. In addition, total phenolic content and antioxidant activity showed a significant increase of extracts were irradiated up to 5 kGy and decrease at dose 7,5 kGy and 10 kGy. Gamma-irradiation up to 10 kGy didn’t affect the activity of ACEi in-vitro while significant difference (P <0.05) of antioxidant activity, total flavonoids and phenolic content on ethanol 70 % extracts P. pellucida L. Kunth herbs. In conclusion, gamma ray irradiation can be used as a preservation method for ethanol 70 % extract P. pellucida L. Kunth.