Background: Low temperature plasma is an ionised gas generated at atmospheric pressure with the temperature of heavy particles (ions, molecules, atoms) close to room temperature. Recent progress highlighted the efficiency of plasma to induce cell death in many cancer cell lines. In the current context of the emergence of the use of cold plasma for medical purposes, this article aims to study the effect of a cold plasma of helium on ex-vivo prostate cells. Methods: A cold atmospheric plasma torch was developed to treat different tumor cells. Parameters like electric voltage, gas flow, cell/torch distance, and exposure time were studied to improve the effects of plasma onto two human prostate tumor cells lines, with different metastatic potential, LNCaP and PC3 and then, compared to human prostate epithelial cells P69. The plasma torch operated by a voltage up to 10 kV, without any risk of arcing, exhibited a visible plasma plume up to about 70 mm, with a temperature close to room temperature. The induced temperature rise due to the plasma treatment is measured to ensure that observed results are not thermal effects. After plasma treatment, microscopy and MTT analyses are achieved to measure cells viability. An ordinary one-way ANOVA test were used between two groups with p-value less than 0.05. Results: An optimized protocol leads to the tumor cells death with regard to cell viability. Cold plasma treatment exhibits differential effect, regarding tumor status of cells without any significant influence of thermal effect (temperature increases of only few degrees Celcius above room temperature). The results show that non-tumor cells P69 are less sensitive to cold plasma exposure with a cell viability of 67% after 120 s of treatment, compared to tumor cell lines LNCaP and PC3, respectively at cell viability of 29% and 23% for the same treatment time. Conclusion: The cold helium plasma treatment showed a selective effect on prostate cells, with an enhanced apoptosis way for tumor cells, whereas the healthy cells are more resistant to the treatment. Moreover, during the treatment, the surface temperature rises only few degrees Celcius. This result is very promising for future in-vivo treatments.