Microproteins and microOpen Reading Frames (microORFs) are small proteins encoded by short, often overlooked, coding regions in the genome. These peptides are typically under 100 amino acids in length and are believed to play critical roles in cellular processes such as stress response, metabolic regulation, and protein quality control. However, their discovery has been hindered by limitations in traditional proteomics and gene annotation pipelines. In this chapter, we present a novel method for the systematic identification of microproteins and microORFs across a range of eukaryotic species using an integrative approach that combines ribosome profiling, mass spectrometry-based proteomics, and advanced bioinformatics tools. Our method enhances the sensitivity of microprotein detection and allows for the validation of candidate microORFs in vivo. This methodology provides a comprehensive platform for microprotein discovery, offering new insights into gene regulation and cellular function.