Protein misfolding is cytotoxic and the accumulation of misfolded proteins threatens cell fitness and viability. Failure to eliminate these polypeptides has been associated with numerous diseases including neurodegenerative disorders. The ubiquitin proteasome system is a major pathway that degrades in the cell these unwanted proteins targeted by protein quality control. Several distinct protein quality control degradation pathways that employ different ubiquitin ligases have been discovered in recent years. Here, we present two novel protein quality control degradation pathways that require the ubiquitin ligases Hul5 and Rsp5 to target cytosolic misfolded proteins for degradation. We used quantitative mass spectrometry to determine that in Saccharomyces cerevisiae, heat-shock triggered a large increase in the level of ubiquitylation of mainly cytosolic proteins. We discovered that the Hul5 ubiquitin ligase participated in this ubiquitylation response. Hul5 was required to maintain cell fitness after heat-shock and to degrade short-lived misfolded proteins. In addition, the localization of Hul5 in the cytoplasm was important for its quality control function. We also showed that Hul5 targeted low-solubility cytosolic proteins in both heat-shock and unstressed conditions. These data indicate that Hul5 is involved in the degradation of cytosolic misfolded proteins. Beside the Hul5 pathway, we found that Rsp5 ubiquitin ligase also participated in the increase of ubiquitylation levels upon heat-shock. Our results indicated that Rsp5 employed a bipartite recognition mechanism to ubiquitylate heat-induced cytosolic targets via the interaction with the Hsp40 co-chaperone Ydj1 and the PY-motifs primarily found in structured regions of these proteins. Notably, we also found that the Rsp5-dependent pathway was dependent on both Ubp2 and Ubp3 deubiquitinases, which acted to mainly reduce the levels of K63-linked ubiquitin chains conjugated to cytosolic misfolded proteins upon heat-shock. The absence of either deubiquitinase led to reduced cell fitness under stress conditions underscoring the importance of the Rsp5-dependent pathway. All together, we identified the two major yeast ubiquitin ligases that mediated the increase in ubiquitylation of cytosolic misfolded proteins upon heat stress. Our work shed new light on protein quality control and how the cell can mediate the degradation of misfolded proteins.