Abstract Although the overall incidence of head and neck squamous cell cancers have decreased in the past 30 years, the incidence oropharyngeal squamous cell carcinoma (OPSCC) related to human papillomavirus (HPV) has actually been rising. Clinically, HPV-related tumors show improved response to treatment and longer overall survival. Recent genomic sequencing data also suggests that HPV-related tumors represent a biologically distinct entity, harboring significantly fewer mutations than non-HPV-related head and neck tumors. The paucity of genetic alterations in HPV-related OPSCC has limited our understanding of its underlying biology and our ability to identify targetable pathways for therapy. Thus, we sought to use RNA sequencing to study post-transcriptional changes in HPV-related OPSCC, primarily alternative splicing events (ASEs). ASEs have been shown to play an important role in carcinogenesis in other cancer types leading to protein expression changes in the absences of genetic alterations at the DNA level. RNA sequencing data was obtained using an Illumina platform in a cohort of 47 HPV-related OPSCC primary tumors and 25 normal mucosa tissue samples from uvulopalatopharyngoplasty surgical specimens. We developed a novel algorithm for the identification of ASEs which utilized outlier analysis to identify differential expression of these splicing events in tumors compared to normal tissues. Our analysis identified 3522 putative alternative splicing events occurring in tumor samples. These ASEs were ranked based on presence of outlier over-expression and under-expression in tumors. The top 200 candidates for over-expression and under-expression were selected for further validation. Differential expression of these candidate ASEs was confirmed using integrated genome viewer (IGV). Additional validation was performed using RT-PCR in the original tumor and normal tissue cohort. These were further validated in publically available TCGA RNA sequencing data. An independent cohort of normal and tissue samples was collected, and differential ASE expression was confirmed using RT-PCR. These methods identified several ASEs that offer insight into the molecular biology of HPV-related OPSCC. This pipeline represents a robust and validated algorithm for identification and prioritizing biologically significant ASEs using RNA sequencing data that may be adapted for analysis in other tumor types. Citation Format: Theresa Guo, Daria Gaykalova, Michael Considine, Justin A. Bishop, William H. Westra, Zubair Khan, Elana Fertig, Joseph A. Califano. Discovery of novel alternative splicing events in human papillomavirus-related oropharyngeal squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4807. doi:10.1158/1538-7445.AM2015-4807