Tumor-specific neoepitopes are promising targets in cancer immunotherapy. However, the identification of functional tumor-specific neoepitopes remains challenging. In addition to the most common source, single-nucleotide variants (SNV), alternative splicing (AS) represents another rich source of neoepitopes and can be utilized in cancers with low SNVs such as uveal melanoma (UM). UM, the most prevalent adult ocular malignancy, has poor clinical outcomes due to a lack of effective therapies. Recent studies have revealed the promise of harnessing tumor neoepitopes to treat UM. Previous studies have focused on neoepitope targets associated with mutations in splicing factor 3b subunit 1 (SF3B1), a key splicing factor; however, little is known about the neoepitopes that are commonly shared by patients independent of SF3B1 status. To identify the AS-derived neoepitopes regardless of SF3B1 status, we herein used a comprehensive nanopore long-read-sequencing approach to elucidate the landscape of AS and novel isoforms in UM. We also performed high-resolution mass spectrometry to further validate the presence of neoepitope candidates and analyzed their structures using the AlphaFold2 algorithm. We experimentally evaluated the antitumor effects of these neoepitopes and found they induced robust immune responses by stimulating interferon (IFN)γ production and activating T cell–based UM tumor killing. These results provide novel insights into UM-specific neoepitopes independent of SF3B1 and lay the foundation for developing therapies by targeting these actionable neoepitopes.