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The rhomboid protein superfamily has been implicated in many important cellular functions including growth factor signaling, protein quality control, mitochondrial homeostasis, and parasite invasion. There are two primary subclasses of the rhomboid superfamily: rhomboid proteases, intramembrane serine proteases that cleave misfolded membrane proteins, and rhomboid pseudoproteases, catalytically inactive proteases. The disposal of misfolded proteins is essential as the accumulation of misfolded proteins causes aggregation, which can become precursors for many neurodegenerative diseases and cancer. Two protein quality control pathways associated with the rhomboid proteins help alleviate this cellular stress, 1) Endoplasmic Reticulum Associated Degradation (ERAD) degrades misfolded proteins while, 2) chaperones reconfigure the folding of misfolded proteins. Dfm1, a yeast rhomboid pseudoprotease, is essential in the degradation of misfolded membrane proteins through ERAD in yeast and regulates sphingolipid homeostasis. Additionally, RHBDL4, a mammalian rhomboid protease, is implicated in breast cancer progression with increased RHBDL4 activity resulting in decreased apoptosis and increased proliferation of breast cancer cells. This thesis describes our original research in further characterizing the function of the rhomboid proteins Dfm1 and RHBDL4. Dfm1, a yeast rhomboid pseudoprotease, is essential in the degradation of misfolded membrane proteins through ERAD in yeast. We establish that Dfm1 has a chaperone-like activity independent from Dfm1’s retrotranslocation role in ERAD. Additionally, we establish another independent and novel role of Dfm1 in the sphingolipid biosynthesis pathway. Here, Dfm1 facilitates sphingolipid homeostasis by exporting phosphorylated Orm2 from the ER which results in the degradation of Orm2 through Endosome and Golgi-Associated Degradation (EGAD). We have also begun to establish a novel in vivo split nano-luciferase assay to interrogate the proteolytic activity of RHBDL4, a mammalian rhomboid protease. RHBDL4 is implicated in breast cancer progression with increased RHBDL4 activity resulting in decreased apoptosis and increased proliferation of breast cancer cells. By utilizing a split-nanoluciferase reporter recognized by RHBDL4, cleavage activity is measured where high luminescence represents RHBDL4 inhibition, and no luminescence indicates RHBDL4 cleavage. The development of this split nano-luciferase assay can then test the efficacy of different RHBDL4 inhibitors and identify a potential therapeutic option.