An in vitro study was conducted to compare the interaction of two cold atmospheric plasma devices on mammalian cells. The two devices were powered by the same FDA-cleared radio frequency power supply, but operated under different power regimes-direct and afterglow. Cells were plasma-treated for various amounts of time, and cell counts were performed at 24 and 48 hours after treatment. Thermal and electrical analyses in combination with chemical analysis of peroxide, nitrites, nitrates, and pH were conducted to identify the long-lived interactions that could impact cell viability. Optical emission spectroscopy was used to monitor the short-lived plasma species generated by each method. It was found that both plasma modes gave rise to differences in the levels of active species generated and this impacted the cellular response.