Small open reading frame-encoded peptides (SEPs) are translated from genome sequences previously unannotated, and they are now recognized as key participants in many physiological and pathological processes. Although ribosome profiling and bioinformatics analysis could predict a large number of SEPs, mass spectrometry (MS) is the only method for their direct 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 investigations. In this study, we successfully developed an effective ammonium formate-mediated C8 solid-phase enrichment (AmF-C8-SPE). Compared to the traditional enrichment approach of Classic-C8, AmF-C8-SPE identified much more SEPs with a higher ratio of unique peptides and a boosted sequence coverage. Utilizing AmF-C8-SPE for 18 pairs of glioma tumors and adjacent normal tissues, we discovered 549 novel SEPs, and 113 of them exhibited differential expression. Importantly, randomly selected SEPs were validated by MS spectral comparison with synthetic peptides and by confirming fusion protein expression in the cells. Furthermore, Mfuzz clustering and ROC curve analyses revealed the SEPs associated with glioma progression. Finally, DeepLoc analyses predicted subcellular locations of these 549 SEPs, two of which were experimentally confirmed in the nucleus by confocal microscopy. Therefore, this study presents an effective SEP identification approach and profiles SEPs in glioma for the first time, offering valuable resources to discover glioma biomarkers/therapeutic targets.