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Background: With the anti-cancer efficacies of cold atmospheric plasma being increasingly recognized in vitro, a demand on creating an effective tool feasible for in vivo animal treatment has emerged. Methods: Through the use of co-axial needles with different calibers in diameter, we designed a novel in situ ejection source of cold atmospheric plasma, namely invivoPen, for animal experiments. It punches just a single pinhole that could considerably ease the complexity of operating with small animals such as mouse. Results: We showed that invivoPen could deliver similar efficacies as plasma activated medium with reduced cost in suppressing cell proliferation and migration as well as potentially boosting the viabilities of mice receiving invivoPen treatment. Blood test, renal and liver functionalities tests all suggest that physical plasma could effectively return tumor-carrying mice to the healthy state without harm to body conditions, and invivoPen slightly outweighs PAM in boosting animal immunity and reducing inflammation. Conclusion: Our study contributes to the community in providing a minimal invasive in situ plasma source, having partly explained the efficacies of cold atmospheric plasma in treating triple negative breast cancers, and proposing the potential synergies between physical plasma and conventional drugs for cancer treatment.