Plasma treatment changes the surface morphology and transforms the surface energy. Therefore, it was applied before the underfill (UF) process of the flip chip to improve the wettability and adhesion of the UF and to prevent delamination or voids. This study focuses on the plasma effects of the regions beneath the flip chip, which comprise the widely used plasma gases, i.e., Ar, N2, and O2. The effects of the plasma treatments are analyzed using atomic force microscopy, X-ray photoelectron spectroscopy, and Dyne ink tests. The surface roughness of the substrate (SBT) center regions is increased from 183.9 nm (bare SBT) to 232.8 nm after the O2 plasma treatment, and the surface roughness of the die center regions is increased from 14.5 to 17.2 nm. The surface-area difference percentage also increases from 47.4% to 61.7% for the SBT center region and from 24.0% to 34.0% for the die center region. The resultant surface energy also increases from 60 to 84 mN/m for the SBT center region and from 66 to 84 mN/m for the die center region. Based on these results, it can be stated that O2 plasma is more effective than other gas plasmas for surface activation, especially beneath the die region.