Non-thermal atmospheric plasma jet (NTAPPJ) is believed to be effective in treating monolayer cells cultured in-vitro due to the oxidative stresses induced. However, it is controversial whether studying the impact of plasma to cells in monolayer can be representative to a model close to the in-vivo tissues. This study revealed and compared the effects of NTAPPJ treatment directly to both monolayer of HeLa cells and microspheroids in the culture media, at different exposure time from 15 to 60 seconds at a fixed power of 80 W. Optical emission spectroscopy (OES) revealed that the plasma applied to the cells is rich in hydroxyl, nitrogen, oxygen and argon species. The experimental results revealed that the treatment time from 15 seconds onwards was detrimental to the monolayer of cells, but not to the spheroids compared with the negative controls. After long NTAPPJ exposure, the HeLa cells in the spheroids were found viable, regardless of the plasma exposure time applied. The multicellular structure of the spheroids has prevented the penetration of free radicals into the cells encapsulated in the spheroids.