Introduction: Peperomia pellucida L. Kunth has been reported to have some biological activities such as antihypertensive and antioxidants. Herbal materials susceptible to contamination during processing and storage which can shortens their shelf life. Gamma-irradiation has been used as a method for preservation. Methods: This research aimed to analyze the effect of gamma-irradiation on inhibition activity of angiotensin converting enzyme (ACE), antioxidant activity, total phenol content, total flavonoid, and thin layer chromatography profiles of P. pellucida L. Herbs extract. The extract was irradiated with 60Co gamma rays at 2.5; 5; 7.5; dan 10 kGy. Results: Irradiation up to 10 kGy did not change ACE inhibitory activity and TLC profile. No significant differences were noted in the inhibition activity of ACE and the type of chromatogram profiles between non-irradiated extract and irradiated extracts up to a dose of 10 kGy 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 ACEinhibitor in-vitro while significant difference (P <0.05) of antioxidant activity, total flavonoids and phenolic content of the extract P. Pellucida. Conclusion: In conclusion, gamma - irradiation can be used as a preservation method for ethanol extract P. Pellucida L. Kunth herbs.