Microproteins, encoded by small open reading frames (smORFs or sORFs) of less than 100 amino acids, play critical roles in gene expression, cellular signaling and metabolism, and hold potential as therapeutic targets. Despite recent genome annotation efforts, many smORFs remain unannotated, leaving a significant gap in our understanding of microprotein structure and function. Efficient microprotein identification is essential for functional studies and improving genome annotation, but remains a technical challenge. Here, we describe a proteogenomic approach that combines microprotein enrichment with mass spectrometry to identify both annotated and novel smORFs. Using this approach, we have successfully identified and validated several novel smORFs in the fully annotated yeast genome. While yeast serves as a model system, this protocol is adaptable to samples from different species, providing a robust framework for microprotein discovery.