Abstract Circulating cell-free DNA (cfDNA) and cell-free RNA (cfRNA) in cancer patients are valuable resource of biomarkers for early detection, diagnosis, treatment and monitoring of disease progression. Compared to cfDNA, cfRNA is more labile to degrade and fluctuate, and thus more challenging to be detected in blood. In the present study, we reported PrediSeq, a validated NGS-based liquid biopsy assay to detect fusions, splicing and mutation events using cfRNA. By using Predicine proprietary ultra-sensitive DeepSeaTM (Deterministic, Efficient, Precise, Sensitive Algorithm) variant detection algorithm, pre-analytical study demonstrated RNA-based detection of fusion, RNA splicing, and mutations using reference materials. In a LOD study, PrediSeq NGS assay is more sensitive than the digital PCR based assay in terms of mutation detection. In clinical validation study, tissue and plasma samples from various types of cancer patients (lung, breast, prostate etc) were used, where concordant fusion events with tissue were observed and RNA mutations confirmed variants detected at DNA level. In conclusion, a highly sensitive and accurate RNA-based liquid biopsy test has been developed to complement the use of cfDNA for detection of fusion, splicing and mutations. Citation Format: Zhixin Zhao. Non-invasive detection of fusion, splicing and mutation events from circulating cell-free RNA in cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3663.