Small open reading frame-encoded peptides (SEPs) are peptides or proteins encoded by previously unannotated sequences in the genome. Although ribosome profiling and bioinformatics could predict a large number of SEPs, mass spectrometry (MS) is the only direct method for their identification. However, MS-based SEP discovery is challenged by the short length and relatively low abundance of SEPs. Thus, there is a critical need to enhance MS approaches for SEP discovery. In this study, we introduce a method based on C8 solid-phase extraction (C8 SPE) with ammonium formate and fractionation (AFF-C8) for the enrichment of SEPs from complex samples. A systematic comparison of Classic-C8, C8-Gel, and AFF-C8 methods revealed that AFF-C8 discovered a greater number of SEPs with enhanced data quality. While C8-Gel improved SEP identification, AFF-C8 demonstrated superior reproducibility. Utilizing AFF-C8 method, we discovered 549 novel SEPs from 18 pairs of glioma tumor and adjacent normal tissues, 113 differentially expressed SEPs. Importantly, 10 SEPs were validated through synthetic peptides, in vitro expression and immunoblotting. Further analysis using Mfuzz clustering and ROC curve filtering comprehensively analyzed SEPs implicated in glioma progression. Finally, DeepLoc predicted the subcellular localization of 549 SEPs, with IP_613981 and SPROHSA206836 localized to the nucleus, a finding confirmed by confocal microscopy. Collectively, this study presents an effective SEP discovery approach and, for the first time, profiles SEPs in glioma, offering a valuable resource for glioma biomarker discovery and functional studies.