The formation of process-related bubbles that become entrapped inside the anisotropic conductive film (ACF) layer during bonding processes remains an issue. The formation of these bubbles is strongly influenced by the process variables, such as bonding pressure and bonding temperature. Therefore, bonding process variables of bonding temperature, bonding pressure, and type of flexible substrate (FS) were changed in order to investigate the effects of the changes as they concern the formation of bubbles. According to the results, the tendency toward bubble formation was closely related to these three factors. The bubble area increased as the bonding temperature increased. Moreover, the shape and tendency of bubbles coincided with temperature distribution in the ACF layer. Two different types of FS, each with different surface roughnesses and energies, were used. The bubbles formed only on the FS with the larger roughness and lower surface energy. According to the results from a surface energy measurement of FS types using goniometry, a FS with a higher surface energy is favorable for a bubble-free assembly, as the higher surface energy provides better wettability. In addition, in order to investigate the effect of bubbles on the reliability of ACF joints, the pressure cooker test (PCT) was performed, and all samples with bubbles electrically failed after 72 h of a PCT, as the process-related bubbles provided a moisture penetration path and entrapment site for moisture. However, all type 1 test vehicles (TVs) survived even after 120 h of a PCT. Therefore, Ar and O-2 plasma treatments were performed on the FS with the lower surface energy in order to improve the surface energies and wettability. Following this, the bubbles were successfully removed at rigid substrate (RS)-FS bonding joints using ACFs.