Human papillomaviruses (HPV) contribute to approximately 5% of all human cancers worldwide, including almost all cervical cancer cases and a growing number of oral cancers. HPV E6 and E7 are the primary viral oncogenes, manipulating host cellular pathways to drive proliferation and survival. The expression of these proteins is essential for the survival of HPV+ cancers; however, their regulation at the protein level is poorly understood. Protein ubiquitination is an essential regulatory mechanism that can alter the stability, activity or localisation of many proteins. Deubiquitinases (DUBs), which remove ubiquitin from target substrates, have become an emerging target in cancer therapeutics. To investigate the role of deubiquitinases in HPV16 E7 stability, we developed a two-colour fluorescence assay which combined a bicistronic plasmid containing EGFP-tagged HPV16E7 protein and a mCherry reporter protein, allowing expression of the EGFP-tagged HPV16 E7 protein and mCherry at a 1:1 ratio, independent of transcriptional differences. Decreased expression of E7 will result in a lower EGFP:mCherry ratio, thus allowing quantification of HPV16 E7 stability. Using a DUB siRNA screening library, we assessed E7 protein expression by flow cytometry and high-content microscopy. This identified several DUBs were identified that may play a role in the stability of HPV16 E7, including USP20, USP33, USP37, USP39, CYLD and MYSM1. Our data demonstrate a novel methodology allowing for assessment of HPV16 E7 oncoprotein stability. Further studies will assess the direct role of these DUBs in the regulation of HPV16 E7 stability and their potential oncogenic functions in HPV-associated cancers.