Small open reading frames-encoded peptides (SEPs) are less than or equal to 100 amino acids which play an critical role in maintaining cell homeostasis under stress. Identification of SEPs under stress conditions is conducive to understanding the expression and molecular function of SEPs. Here we used mass spectrometry-based proteomics technology to explore SEPs with potential cellular stress function in Saccharomyces cerevisiae. Finally, 258 microproteins with unique peptides were identified under six culture conditions: normal, oxidation, starvation, UV radiation, heat shock, and heat shock with starvation.