Micropeptides encoded by small open reading frames (sORFs) represent a newly recognized class of biomolecules involved in important cellular functions such as metabolism, signal transduction and homeostasis. In the past, these peptides have been overlooked due to annotation and technical challenges. Recent advances in high-throughput techniques such as ribosome profiling, liquid chromatography-tandem mass spectrometry (LC–MS/MS) and bioinformatics tools have enabled the systematic identification and characterization of these peptides. In prostate cancer (PCa), urinary micropeptides have been shown to be promising noninvasive biomarkers, as the prostate is located close to the urinary tract and urine can be easily collected. In this chapter, we discuss the methods used to detect micropeptides, incorporating in silico predictions, LC–MS/MS-based proteomic analyzes, and metabolic pathway enrichment approaches. We highlight the recent discoveries of micropeptides with differential expression in PCa and their potential as diagnostic and prognostic biomarkers. These findings highlight the importance of micropeptide research and pave the way for new strategies in PCa diagnosis and personalized medicine.