Protein degradation via the proteasome is a fundamental process for maintaining proteostasis. The post-translational modification of substrate proteins by ubiquitin and the ubiquitin-like modifier FAT10 targets them for proteasomal degradation. While ubiquitin and FAT10 have traditionally been perceived as passive signals for proteasomal targeting, emerging evidence indicates that they actively influence both the thermodynamic and conformational landscapes of their respective substrates. In this review, we explore recent mechanistic insights into how the modification site and the intrinsic characteristics of the modifier dictate substrate stability. Ubiquitin destabilizes proteins in a site-specific manner through entropic restriction or enthalpic disruption, thereby modulating degradation efficiency. It is noteworthy that well-folded ubiquitin substrates require unfoldases such as p97/valosin-containing protein for successful degradation. Conversely, FAT10 acts as a significant destabilizer across various substrates due to its inherent low thermodynamic stability and flexible structure, thereby facilitating rapid degradation independent of unfoldases. These findings redefine post-translational tagging as an active regulator of protein fate and propose novel strategies for manipulating protein turnover within disease contexts.