Background: EGFR and ERBB2 exon 20 insertion (exon20ins) driver mutations are well described in non-small cell lung cancer due to their resistance to early generation EGFR tyrosine kinase inhibitors. This has led to the development and ultimate approval of exon20ins specific inhibitors, such as mobocertinib. Previous studies have reported EGFR and ERBB2 exon20ins mutations in 0.15%-1.91% of urothelial carcinoma (UC) patients, but this is likely an underrepresentation as indel variants are often under detected by commonly used NGS-variant analysis tools. Additionally, little is known about the biological consequences of EGFR and ERBB2 exon20ins mutations in UC. In this study, we aimed to: 1) characterize the prevalence of EGFR and ERBB2 exon20ins mutations in UC; 2) assess how EGFR and ERBB2 exon20ins mutations impact malignant transformation; and 3) evaluate the sensitivity of UC cell lines with the exon20ins to mobocertinib. Methods: UC patient datasets were realigned using the indel caller, ABRA2, then EGFR and ERBB2 exon20ins frequencies were calculated. Next, parental KT1970 urothelial cells expressing either EGFR WT, ERBB2 WT, or exon20ins mutation constructs (EGFRinsH [insH] or ERBB2insYVMA [YVMA]) were generated and in vitro transformation assays were performed. Anchorage-independent growth was assessed with soft agar assay. Engineered urothelial organoids with various exon20ins variants were created to assess for their ability to differentiate. Finally, sensitivity to erlotinib and mobocertinib was determined. Results: ABRA2 identified a prevalence of ERBB2 and EGFR Exon20ins mutations in 2.5% (7/275) of urothelial tumors in the IMVigor210 patient cohort. ERBB2 insYVMA expressing organoids had the highest basal marker (KRT5) expression in proliferation media which decreased in differentiation media. In the soft agar assay, YVMA cells had a 9.26 fold increase in colony formation (CF) compared to the ERBB2 WT counterpart and insH had a 6.02 fold increase in CF over the EGFR WT counterpart. The IC50 for mobocertinib (M) was significantly lower than erlotinib (E) in the exon20ins cell lines (insH M: 0.123 uM, E:10.69 uM / YVMA M: 0.57 uM, E: 13.54 uM). The difference in IC50 values for mobocertinib vs erlotinib in the WT cells was less pronounced. Conclusions: The prevalence of EFGR and ERBB2 exon20 insertion mutations is likely unrepresented in urothelial carcinoma by current variant calling pipelines. The ERBB2 YVMA mutation showed a more aggressive phenotype and possible basal pattern in vitro which may have implications in terms of prognosis and treatment considerations. Both EGFR and ERBB2 exon20ins urothelial cells were more sensitive to mobocertinib. This study provides evidence that exon20ins specific inhibitors could offer additional targeted therapy options for patients with urothelial cancer who have EGFRinsH and ERBB2insYVMA mutations.