Plant regulatory peptides represent a novel class of signaling molecules that play a central role in the regulation of plant growth, development, and stress responses. Owing to high biological activity at low concentrations, they are considered promising biostimulants for environmentally sustainable agriculture. This review summarizes the key theoretical approaches and experimental methods used for the discovery and identification of plant regulatory peptides, including mass spectrometry, bioinformatics, bioassays, and in silico screening. An overview is provided of the major plant regulatory peptides identified to date, such as systemin, PSK, PSY, AtPep1, CLV3, TDIF, CEP, and CIF, along with the methods used for their isolation, chemical synthesis, and functional validation. Special attention is given to model systems based on cell cultures and seedlings, which are commonly employed to screen peptide activity, as well as to strategies for identifying their corresponding receptors. The review highlights the critical role of bioassays as a final and indispensable stage in peptide discovery pipelines, enabling the functional evaluation of both identified and putative peptides.